缺氧诱导的circPLOD2a/b通过与HuR结合抑制XIRP1，促进胶质母细胞瘤的侵袭性-动脉网

Hypoxia-induced circPLOD2a/b promotes the aggressiveness of glioblastoma by suppressing XIRP1 through binding to HuR

Nature 等信源发布 2025-01-17 13:28

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Abstract

摘要

Hypoxia is a common feature of glioblastoma (GBM). Circular RNAs (circRNAs) are identified as regulators in cancers. However, the role of circRNAs in GBM remains elusive. Here, circPLOD2a and circPLOD2b, spliced from the same parental gene

。环状RNA（circRNA）被鉴定为癌症中的调节剂。然而，circRNA在GBM中的作用仍然难以捉摸。在这里，circPLOD2a和circPLOD2b是从同一亲本基因剪接而来的

PLOD2

, are identified as hypoxia-responsive circRNAs. Overexpression of circPLOD2a and b enhance while knockdown inhibit GBM cell aggressiveness. The protein partners and downstream molecules were investigated by RNA-pulldown, mass spectrometry and RNA-seq. Mechanistically, HIF1α induces the expression of circPLOD2a and b, which competitively bind to HuR, causing a degradation of .

，被鉴定为缺氧反应性circRNA。circPLOD2a和b的过表达增强，而敲低抑制GBM细胞的侵袭性。通过RNA下拉，质谱和RNA-seq研究了蛋白质伴侣和下游分子。从机制上讲，HIF1α诱导circPLOD2a和b的表达，其竞争性结合HuR，导致降解。

XIRP1

in vitro and in vivo. Clinical data demonstrate circPLOD2a and b are highly expressed in GBM negatively correlated with

体外和体内。

XIRP1

, whose lower expression associates with higher glioma grade and worse prognosis. In conclusion, hypoxia-induced circPLOD2a and b are oncogenic regulators of tumour aggressiveness through attenuating the interaction between HuR and

，其低表达与较高的胶质瘤分级和较差的预后相关。总之，缺氧诱导的circPLOD2a和b通过减弱HuR和HuR之间的相互作用而成为肿瘤侵袭性的致癌调节剂

XIRP1

in glioblastoma cells and may be potential therapeutic targets for this disease.

在胶质母细胞瘤细胞中，可能是该疾病的潜在治疗靶点。

Introduction

简介

Glioblastoma multiforme (GBM) is the highest grade of gliomas and one of the most malignant brain tumors in adults. Although great progresses have achieved in diagnose and therapy, the 5-year overall survival rate of GBM is still <35%

多形性胶质母细胞瘤（GBM）是胶质瘤的最高级别，也是成人中最恶性的脑肿瘤之一。虽然在诊断和治疗方面取得了很大进展，但GBM的5年总生存率仍低于35%

. One major difficulty of surgical removal is the propensity of GBM to infiltrate into adjacent normal brain tissues. Therefore, it is crucial to excavate mechanisms underlying the aggressiveness of GBM and explore new therapeutic targets for this disease. Hypoxia is a common microenvironment in solid tumors including GBM, which is caused by excessive cell proliferation and poorly organized tumor vasculature.

手术切除的一个主要困难是GBM倾向于渗入邻近的正常脑组织。因此，挖掘GBM侵袭性的潜在机制并探索这种疾病的新治疗靶点至关重要。缺氧是包括GBM在内的实体瘤中常见的微环境，这是由过度的细胞增殖和组织不良的肿瘤脉管系统引起的。

. Hypoxia profoundly affects many signaling pathways to induce cellular adaptations

缺氧深刻影响许多信号通路，诱导细胞适应

. HIF1α is considered as the core regulator under hypoxia which activates gene transcription and leads to a more aggressive and metastatic phenotype of cells

HIF1α被认为是缺氧条件下的核心调节因子，可激活基因转录并导致细胞更具侵袭性和转移性的表型

. Knock-down of HIF1α impairs cell invasion and migration in GBM cells

.敲除HIF1α会损害GBM细胞中的细胞侵袭和迁移

. However, previous studies have mainly focused on the coding RNAs regulated by hypoxia and HIF1α. The functions of non-coding RNAs, especially circRNAs under hypoxia has been poorly understood in GBM.

然而，以前的研究主要集中在缺氧和HIF1α调控的编码RNA上。GBM中对非编码RNA，特别是缺氧条件下circRNA的功能了解甚少。

Circular RNA (circRNA) is a type of single-stringed RNA that comes from back-splicing of pre-mRNA with a covalently closed loop structure

环状RNA（circRNA）是一种单链RNA，来自具有共价闭环结构的前mRNA的反向剪接

. The conserved sequences and tumor-specific expression make circRNAs potentially valuable biomarkers for cancer diagnoses. Recent studies indicate that circRNAs are involved in the response to hypoxic microenvironment and the progression of cancers

保守序列和肿瘤特异性表达使circRNA成为癌症诊断的潜在有价值的生物标志物。最近的研究表明，circRNA参与了对缺氧微环境的反应和癌症的进展

. CircWSB1 is reported to be up-regulated by HIF1α and promotes breast cancer progression

据报道，CircWSB1被HIF1α上调并促进乳腺癌的进展

. Another hypoxia-induced exosome circ133 increases cell invasion and migration by acting on miR-133a/GEF-H1/RhoA axis in colorectal cancer

另一种缺氧诱导的外泌体circ133通过作用于结直肠癌中的miR-133a/GEF-H1/RhoA轴来增加细胞侵袭和迁移

. Dysregulated circRNAs are implicated in GBM as miRNA sponges, molecular partners of RBPs or coding sequences for short peptides

失调的circRNA与GBM有关，如miRNA海绵，RBP的分子伴侣或短肽的编码序列

. Nevertheless, the biological function of circRNAs in response to hypoxia in GBM remains largely unknown.

然而，circRNA对GBM缺氧反应的生物学功能仍然很大程度上未知。

Human antigen R (HuR), a member of the embryonic lethal abnormal visual protein (ELAV) family, acts as an RNA binding protein in cell cycle progression

人类抗原R（HuR）是胚胎致死异常视觉蛋白（ELAV）家族的成员，在细胞周期进程中充当RNA结合蛋白

. There are three RNA recognition motifs (RRMs) in HuR that bind to the poly-U elements or AU-rich elements (AREs) in the 3′-untranslated region (3′-UTR) of target genes

HuR中有三个RNA识别基序（RRM），它们与靶基因3'非翻译区（3'-UTR）中的poly-U元件或富含AU的元件（are）结合

. Previous studies have revealed that HuR plays a vital role in tumor oncogenesis, hyperplasia, invasion and migration by regulating the stabilization of a large subset of target mRNAs

先前的研究表明，HuR通过调节大部分靶mRNA的稳定性，在肿瘤的发生，增生，侵袭和迁移中起着至关重要的作用

. For example, HuR stabilizes the mRNA of

例如，HuR稳定了

PTBP1

and promotes lung metastasis of colorectal cancer

并促进结直肠癌的肺转移

. It was also reported that HuR is widely involved in circRNA-mediated signaling regulation

据报道，HuR广泛参与circRNA介导的信号调节

In this study, we identified two hypoxia-induced circRNAs in GBM, derived from the same parental gene

在这项研究中，我们在GBM中鉴定了两个缺氧诱导的circRNA，它们来自同一亲本基因

PLOD2

and termed as circPLOD2a and circPLOD2b, which are regulated by HIF1α. CircPLOD2a/b promote the invasion and migration of GBM cells both in vivo and in vitro. We further identified HuR as the protein partner of circPLOD2a/b by RNA-pull down plus mass spectrometry. Mechanistically, circPLOD2a/b suppress the expression of .

并被称为circPLOD2a和circPLOD2b，它们受HIF1α调节。CircPLOD2a/b在体内和体外促进GBM细胞的侵袭和迁移。我们通过RNA下拉加质谱法进一步将HuR鉴定为circPLOD2a/b的蛋白质伴侣。从机制上讲，circPLOD2a/b抑制的表达。

XIRP1

mRNA through competitive binding with HuR. Our findings provide insights into the involvement of circRNAs in hypoxic response, and suggest that these two circRNAs may be potential biomarkers and therapeutic targets for GBM.

mRNA通过与HuR竞争性结合。我们的研究结果提供了circRNA参与低氧反应的见解，并表明这两种circRNA可能是GBM的潜在生物标志物和治疗靶点。

Results

结果

CircPLOD2a/b are identified as hypoxia-responsive circRNAs in GBM cells

CircPLOD2a/b在GBM细胞中被鉴定为缺氧反应性circRNA

To identify circRNAs which are involved in responses to hypoxia in GBM cells, RNA-seq (GSE245635) was performed in U87 cells cultured under normoxia (21% O

为了鉴定参与GBM细胞缺氧反应的circRNA，在常氧（21%O

) and hypoxia (1% O

)和缺氧（1%O

) for 24 h. Data analysis showed that 17 circRNAs were significantly up-regulated under hypoxia with a cut-off criterion of |log2fold-change | > 1.0 and

)数据分析显示，17个circRNA在缺氧条件下显着上调，截止标准为log2倍变化>1.0和

-value < 0.05 (Fig.

-值>0.05（图。

1A, B

1A、B

). Divergent qRT-PCR primers cross the junction sites of these 17 circRNAs were designed in circBase (

)。在circBase中设计了跨越这17个circRNA连接位点的不同qRT-PCR引物(

http://circbase.org/

) to testify the RNA-seq data. Nine circRNAs were confirmed to be up-regulated in U87 under hypoxic condition (Fig.

)验证RNA-seq数据。在低氧条件下，9个circRNA被证实在U87中上调（图）。

1C级

). The basal expression levels of these 9 circRNAs in hypoxic U87 cells were showed in Fig.

)。缺氧U87细胞中这9种circRNA的基础表达水平如图所示。

1维

. The existence of these circRNAs were further examined by RT-PCR and agarose-gel electrophoresis in U87, and the results showed that all these 9 circRNAs could be amplified with divergent primers in cDNA samples but not in gDNA samples (Fig.

通过RT-PCR和琼脂糖凝胶电泳在U87中进一步检测了这些circRNA的存在，结果表明，所有这9个circRNA都可以在cDNA样品中用不同的引物扩增，但在gDNA样品中不能扩增（图）。

1E级

and Supplementary Fig.

和补充图。

8A个

). Considering both the alteration and abundance of these circRNAs in U87 cells (Fig.

)。考虑到U87细胞中这些circRNA的改变和丰度（图）。

1C–E

), hsa_circ_0141963 and hsa_circ_0067682 were chosen as top candidates for further investigation.

)，hsa\u circ\u 0141963和hsa\u circ\u 0067682被选为进一步调查的最佳候选人。

Fig. 1: CircPLOD2a/b are up-regulated in GBM cells under hypoxia.

图1:CircPLOD2a/b在缺氧条件下在GBM细胞中上调。

Heatmap shows differentially expressed circRNAs between normoxia and hypoxia in U87 cells on a scale from blue (downregulated,

热图显示U87细胞中常氧和缺氧之间差异表达的circRNA，从蓝色（下调，

= 2158) to red (upregulated,

2158）变为红色（上调，

= 2566).

（2566）

B类

Volcano plot shows differently expressed circRNAs between normoxia and hypoxia in U87 cells,

火山图显示U87细胞中常氧和缺氧之间不同表达的circRNA，

= 4727. Horizontal dashed line labels

=4727。水平虚线标签

= 0.05. Vertical dashed lines label |log2fold-change | = 1.

= 0.05。垂直破折线标签 |log2fold-change | = 1。

C级

Verification of 17 up-regulated circRNAs (

验证17个上调的circRNA(

-value < 0.05 and reads counts >10) by qRT-PCR,

-，

= 3.

= 3。

Basal expression levels of 9 up-regulated circRNAs in U87 cells under hypoxia examined by qRT-PCR,

通过qRT-PCR检测缺氧条件下U87细胞中9种上调circRNA的基础表达水平，

= 3.

Verification of corresponding circRNAs in U87 cells by RT-PCR using divergent and convergent primers.

使用发散和会聚引物通过RT-PCR验证U87细胞中相应的circRNA。

F级

Schematic illustration of the generation of circPLOD2a/b.

产生circPLOD2a/b的示意图。

克

Junction sites of circPLOD2a/b were validated by Sanger sequencing.

通过Sanger测序验证了circPLOD2a/b的连接位点。

Relative levels of circPLOD2a/b after treated with or without RNase R digesting determined by qRT-PCR in U87 cells,

通过qRT-PCR在U87细胞中测定经或不经RNase R消化处理后circPLOD2a/b的相对水平，

= 3.

= 3。

我

Relative expression levels of (

(

我

) circPLOD2a and (

)circPLOD2a和(

) circPLOD2b under normoxia and hypoxia in GBM cell lines determined by qRT-PCR,

)通过qRT-PCR测定的GBM细胞系中常氧和缺氧条件下的circPLOD2b，

= 3.

= 3。

Cellular localization of circPLOD2b examined by FISH with a junction specific antisense probe (red). Scale bar: 25 μm.

用连接特异性反义探针（红色）通过FISH检查circPLOD2b的细胞定位。比例尺：25μm。

U87 cells were treated for 0 h, 12 h, 24 h, 48 h under 1% O

U87细胞处理0小时，12小时，24小时，1%O下48小时

. Protein level of HIF1α and expression level of circPLOD2a/b were verified by western blot and qRT-PCR respectively,

western blot和qRT-PCR分别检测HIF1α蛋白水平和circPLOD2a/b表达水平，

= 3.

= 3。

Protein level of HIF1α and expression level of circPLOD2a/b in (

HIF1α的蛋白水平和circPLOD2a/b的表达水平(

) OE-HIF1α and (

OE-HIF1α 和

) KD-HIF1α U87 cells under hypoxia, n = 3. Relative integrated density normalized to β-actin was marked above each band. Data are shown as mean ± SEM (error bars) and were analyzed using Student’s

)缺氧条件下的KD-HIF1αU87细胞，n=3。相对于β-肌动蛋白标准化的相对整合密度标记在每个条带上方。数据显示为平均SEM（误差线），并使用学生的

-test (

-测试(

) and ANOVA (

)和方差分析(

C级

–

). NA, not available.

)。不适用，不可用。

Full size image

全尺寸图像

Referring to the position information in circBase, hsa_circ_0141963 and hsa_circ_0067682 were spliced from the exon 2-3 and exon 2–7 of the same parental gene

参考circBase中的位置信息，从同一亲本基因的外显子2-3和外显子2-7剪接了hsa\u circ\u 0141963和hsa\u circ\u 0067682

PLOD2

, respectively (Fig.

，分别（图）。

1层

). Thus, we named hsa_circ_0141963 as circPLOD2a and hsa_circ_0067682 as circPLOD2b. The results of sanger sequencing showed continuous signals at the junction sites of both circPLOD2a and circPLOD2b (Fig.

)。因此，我们将hsa\u circ\u 0141963命名为circPLOD2a，将hsa\u circ\u 0067682命名为circPLOD2b。桑格测序的结果显示在circPLOD2a和circPLOD2b的连接位点都有连续的信号（图）。

1克

). Besides, circPLOD2a/b could be amplified after RNase R digesting, while negative controls, linear

)。此外，RNase R消化后可以扩增circPLOD2a/b，而阴性对照则是线性的

PLOD2

and

和

β-actin

β-阿克汀

, showed a significant decrease (Fig.

，显示出显着的下降（图）。

1小时

). Furthermore, we examined the expression of circPLOD2a/b under hypoxia in other GBM cell lines except for U87. Both circPLOD2a and circPLOD2b could be significantly induced by hypoxia in all the tested GBM cell lines including DBTRG, LN229, T98G and U251 (Fig.

)。此外，我们检测了除U87外的其他GBM细胞系中缺氧条件下circPLOD2a/b的表达。在所有测试的GBM细胞系（包括DBTRG，LN229，T98G和U251）中，缺氧均可显着诱导circPLOD2a和circPLOD2b（图）。

1I, J

1I，J

). RNA FISH assay was conducted to explore the cellular localization of circRNA2b in U87 cells. A FISH probe mix targeting circPLOD2b junction site were synthesized and incubated with U87 cells. The results showed that circPLOD2b mainly located in cytoplasm, and was significantly induced by hypoxic condition (1% O.

)。。合成靶向circPLOD2b连接位点的FISH探针混合物，并与U87细胞孵育。结果表明，circPLOD2b主要位于细胞质中，低氧条件（1%O。

, 48 h) (Fig.

，48小时）（图）。

1公里

), which was consistent with the results of qRT-PCR. We were not able to perform FISH on circPLOD2a due to lacking of specific probes.

)，这与qRT-PCR的结果一致。由于缺乏特异性探针，我们无法对circPLOD2a进行FISH。

Our findings suggest that circPLOD2a and circPLOD2b are hypoxia-induced circRNAs and probably involved in the responses to hypoxia in GBM cells.

我们的研究结果表明，circPLOD2a和circPLOD2b是缺氧诱导的circRNA，可能参与GBM细胞对缺氧的反应。

CircPLOD2a/b are regulated by HIF1α under hypoxia

HIF1α is a key regulator of hypoxic responses. Previous studies showed that linear

HIF1α是低氧反应的关键调节剂。先前的研究表明，线性

PLOD2

was regulated by HIF1α in hypoxic condition to promote tumor invasion and progression

在低氧条件下受HIF1α调节，以促进肿瘤的侵袭和进展

. To investigate the regulation of HIF1α on circPLOD2a/b, we cultured U87 cells under hypoxic conditions for 12 h, 24 h, and 48 h. The expression of circPLOD2a/b along with HIF1α protein level, were both gradually increased in a time-dependent manner (Fig.

为了研究HIF1α对circPLOD2a/b的调节，我们在低氧条件下培养U87细胞12小时，24小时和48小时。circPLOD2a/b的表达以及HIF1α蛋白水平均以时间依赖性方式逐渐增加（图）。

1升

and Supplementary Fig.

和补充图。

8B个

). Then we established stable cell lines with HIF1α overexpression or knockdown in U87 and cultured the cells under hypoxic condition. The overexpression of HIF1α led to elevation of both circPLOD2a and circPLOD2b (Fig.

)。。HIF1α的过度表达导致circPLOD2a和circPLOD2b的升高（图）。

1百万

and Supplementary Fig.

和补充图。

8摄氏度

). In contrast, circPLOD2a/b showed a significant decrease when HIF1α was knocked down (Fig.

)。相反，当HIF1α被敲除时，circPLOD2a/b显示出显着降低（图）。

1牛

and Supplementary Fig.

和补充图。

Linear

线性

PLOD2

has been reported to be upregulated in hypoxic cancer cells, including GBM

据报道，在包括GBM在内的缺氧癌细胞中上调

. Our data suggest that HIF1α enhances the transcription of the

我们的数据表明HIF1α增强了

PLOD2

gene. Analysis using the JASPAR database (

基因。使用JASPAR数据库进行分析(

https://jaspar.elixir.no/

) predicted 13 potential HIF1α binding sites within the 0–2000 bp promoter region of

)

PLOD2

, with a relative profile score threshold of 80%. Accordingly, dual-luciferase reporter assays demonstrated that transfection with the HIF1α overexpression plasmid significantly elevated the luciferase activity of the

，相对概况得分阈值为80%。因此，双荧光素酶报告基因检测表明，用HIF1α过表达质粒转染可显着提高荧光素酶的活性

PLOD2

promoters (Supplementary Fig.

启动子（补充图）。

1C级

). These data demonstrated that the expression of circPLOD2a/b is notably regulated by HIF1α under hypoxia.

)。这些数据表明circPLOD2a/b的表达在缺氧条件下受到HIF1α的显着调节。

CircPLOD2a/b promote invasion and migration of GBM cells under hypoxia in vitro

CircPLOD2a/b在体外缺氧条件下促进GBM细胞的侵袭和迁移

To explore the functions of circPLOD2a/b in GBM cells, we inspected the effects of overexpression and knockdown of these two circRNAs on the proliferation, invasion and migration of GBM cells. U87 and LN229 cell lines, which possess relatively high expression levels of circPLOD2a/b under hypoxia (Fig. .

为了探索circPLOD2a/b在GBM细胞中的功能，我们检查了这两种circRNA的过表达和敲低对GBM细胞增殖，侵袭和迁移的影响。U87和LN229细胞系，在缺氧条件下具有较高的circPLOD2a/b表达水平（图）。

1I, J

1I，J

; Supplementary Fig.

。

1A, B

1A、B

), were employed to establish stable cell lines of circPLOD2a/b knockdown and overexpression. CircPLOD2a/b were knocked down by shRNAs targeting the back-splicing region. The mRNA and protein levels of linear

)，用于建立稳定的circPLOD2a/b敲低和过表达细胞系。CircPLOD2a/b被靶向反向剪接区域的shRNA敲低。线性的mRNA和蛋白质水平

PLOD2

were not affected by the manipulations of circPLOD2a/b in GBM cells (Supplementary Fig.

不受GBM细胞中circPLOD2a/b操作的影响（Supplementary Fig.）。

). The results of CCK-8 assay showed no significant differences in U87 and LN229 cells when circPLOD2a or circPLOD2b was overexpressed (Fig.

)。当circPLOD2a或circPLOD2b过表达时，CCK-8测定的结果显示U87和LN229细胞没有显着差异（图）。

2E, F, M, N

2E、F、M、N

), indicating that circPLOD2a/b had no significant influence on the cell viability of GBM cells under hypoxia.

)，表明circPLOD2a/b对缺氧条件下GBM细胞的细胞活力没有显着影响。

Fig. 2: Overexpression of circPLOD2a/b promotes GBM cell invasion and migration under hypoxia.

图2:circPLOD2a/b的过表达促进缺氧条件下GBM细胞的侵袭和迁移。

B类

Results of transwell assays in (

transwell分析结果(

) U87 and (

)U87和(

B类

) LN229 OE-circPLOD2a cells under hypoxia. Scale bar: 100 μm.

)缺氧条件下的LN229 OE-circPLOD2a细胞。比例尺：100微米。

C级

Quantification of transwell assays in (

(

C级

) U87 and (

)U87和(

) LN229 OE-circPLOD2a cells,

)LN229 OE-circPLOD2a细胞，

= 3.

= 3。

F级

Results of CCK8 assays on (

CCK8检测结果(

) U87 and (

)U87和(

F级

) LN229 OE-circPLOD2a cells under hypoxia,

)缺氧条件下的LN229 OE-circPLOD2a细胞，

= 5.

= 5。

克

小时

Representative pictures and quantification of wound-healing assays in (

伤口愈合测定的代表性图片和量化(

克

) U87 and (

)U87和(

小时

) LN229 OE-circPLOD2a cells under hypoxia. Scale bar: 250 μm,

)缺氧条件下的LN229 OE-circPLOD2a细胞。比例尺：250微米，

= 5.

= 5。

我

Results of transwell assay in (

transwell分析结果(

我

) U87 and (

)U87和(

) LN229 OE-circPLOD2b cells under hypoxia. Scale bar: 100 μm.

)缺氧条件下的LN229 OE-circPLOD2b细胞。比例尺：100微米。

Quantification of transwell assays in (

(

) U87 and (

)U87和(

) LN229 OE-circPLOD2b cells,

)LN229 OE-circPLOD2b细胞，

= 3.

= 3。

Results of CCK8 assay on (

CCK8检测结果(

) U87 and (

)U87和(

) LN229 OE-circPLOD2b cells,

)LN229 OE-circPLOD2b细胞，

= 5.

= 5。

Representative pictures and quantification of wound-healing assay (

伤口愈合测定的代表性图片和定量(

) U87 and (

)U87和(

) LN229 OE-circPLOD2b cells under hypoxia,

)缺氧条件下的LN229 OE-circPLOD2b细胞，

= 5. Scale bar: 250 μm. Data are shown as mean ± SEM (error bars) and analyzed using Student’s

比例尺：250微米。数据显示为平均SEM（误差线），并使用学生的

test (

测试(

C级

–

) and ANOVA (

)和方差分析(

–

Full size image

全尺寸图像

Transwell and wound healing assays were conducted to evaluate the functions of circPLOD2a and circPLOD2b in GBM cell invasion and migration under hypoxia. When circPLOD2a (Fig.

进行Transwell和伤口愈合测定以评估circPLOD2a和circPLOD2b在缺氧条件下GBM细胞侵袭和迁移中的功能。当circPLOD2a（图。

2A–D, G, H

2A–D、G、H

) or circPLOD2b (Fig.

)或circPLOD2b（图）。

2I–L, O, P

2I–L、O、P

) was stably overexpressed in U87 and LN229 cells, the cell invasion and migration ability significantly increased under hypoxia. On the other side, when circPLOD2a (Fig.

)在U87和LN229细胞中稳定过表达，在缺氧条件下细胞侵袭和迁移能力显着增加。。

3A–D

) or circPLOD2b (Fig.

)或circPLOD2b（图）。

3E–H

) was knocked down, the cell invasion and migration ability under hypoxia was significantly inhibited.

)被击倒，缺氧条件下细胞的侵袭和迁移能力受到显着抑制。

Fig. 3: Knockdown of circPLOD2a/b inhibits GBM cell invasion and migration under hypoxia.

图3:circPLOD2a/b的敲低在缺氧条件下抑制GBM细胞的侵袭和迁移。

B类

Results of transwell assays in (

transwell分析结果(

) U87 and (

)U87和(

B类

) LN229 KD-circPLOD2a cells under hypoxia. Scale bar: 100 μm,

)缺氧条件下的LN229 KD-circPLOD2a细胞。比例尺：100微米，

= 3.

= 3。

C级

Results of wound-healing assays in (

伤口愈合试验结果(

C级

) U87 and (

)U87和(

) LN229 KD-circPLOD2a cells under hypoxia. Scale bar: 250 μm,

)缺氧条件下的LN229 KD-circPLOD2a细胞。比例尺：250微米，

= 5.

= 5。

F级

Results of transwell assays in (

transwell分析结果(

) U87 and (

)U87和(

F级

) LN229 KD-circPLOD2b cells under hypoxia. Scale bar: 100 μm,

)缺氧条件下的LN229 KD-circPLOD2b细胞。比例尺：100微米，

= 3.

= 3。

克

小时

Results of wound-healing assays in (

伤口愈合试验结果(

克

) U87 and (

)U87和(

小时

) LN229 KD-circPLOD2b cells under hypoxia. Scale bar: 250 μm,

)缺氧条件下的LN229 KD-circPLOD2b细胞。比例尺：250微米，

= 5. Data are shown as mean ± SEM (error bars) and analyzed using ANOVA. Scr: Scramble.

数据显示为平均值±SEM（误差线），并使用ANOVA进行分析。Scr：加扰。

Full size image

全尺寸图像

As a summary, our results demonstrate that circPLOD2a and circPLOD2b function to promote GBM cell invasion and migration under hypoxia in vitro.

总之，我们的研究结果表明，circPLOD2a和circPLOD2b在体外缺氧条件下促进GBM细胞的侵袭和迁移。

CircPLOD2a/b directly interact with HuR

CircPLOD2a/b直接与HuR交互

To explore the protein partners of circPLOD2a/b, RNA pull-down assays were performed with biotin-labeled probes targeting the back-splicing site of circPLOD2a (Fig.

为了探索circPLOD2a/b的蛋白质伴侣，用靶向circPLOD2a反向剪接位点的生物素标记探针进行RNA下拉测定（图）。

4A, B

4A，B

) and circPLOD2b (Fig.

)和circPLOD2b（图）。

4C, D

4C，D

) in hypoxic U87 cells. Mass spectrometry identified 69 proteins which were pulled down by antisense probe targeting circPLOD2a and 53 proteins by probe targeting circPLOD2b, but not by the scramble probe. Crossing analysis with the established RBP database (

)在缺氧的U87细胞中。质谱鉴定了69种蛋白质，这些蛋白质被靶向circPLOD2a的反义探针拉下，53种蛋白质被靶向circPLOD2b的探针拉下，但没有被扰乱探针拉下。与已建立的RBP数据库进行交叉分析(

http://rbpdb.ccbr.utoronto.ca

) and the cancer metastasis-related RBPs revealed by previous studies

)以及先前研究揭示的与癌症转移相关的RBP

determined five potential interacting partners of circPLOD2a or circPLOD2b (Fig.

确定了circPLOD2a或circPLOD2b的五个潜在相互作用伙伴（图）。

4B, D

4B，D

). Further validation of RNA pull-down with circPLOD2a/b probes and corresponding antibodies confirmed the interaction of circPLOD2a/b with HuR (Fig.

)。用circPLOD2a/b探针和相应抗体进一步验证RNA下拉，证实了circPLOD2a/b与HuR的相互作用（图）。

4E, F

4E，F

, Supplementary Fig.

，补充图。

9A, B

9A，B

). RIP assay also disclosed the endogenous binding of circPLOD2a/b, but not linear

)。RIP分析还揭示了circPLOD2a/b的内源性结合，但不是线性的

PLOD2

with HuR (Fig.

与HuR（Fig.

4G–J

and Supplementary Fig.

和补充图。

9摄氏度

). FISH-IF assay revealed that circPLOD2b and HuR co-localize in the cytoplasm of U87 cells. A stronger binding was observed in U87 cells under hypoxic conditions compared to normoxic conditions (Fig.

)。。与常氧条件相比，在低氧条件下U87细胞中观察到更强的结合（图）。

Fig. 4: CircPLOD2a/b bind to HuR in U87 cell.

图4:CircPLOD2a/b与U87细胞中的HuR结合。

–

Proteins pulled down by biotin-labeled circPLOD2a (

生物素标记的circPLOD2a下拉的蛋白质(

) or circPLOD2b (

)或circPLOD2b(

C级

) antisense probes and corresponding scrambled control probes in hypoxic U87 cells were visualized by SDS-PAGE and silver staining. Unique peptides identified by mass spectrometry in (

)通过SDS-PAGE和银染观察缺氧U87细胞中的反义探针和相应的乱序对照探针。通过质谱鉴定的独特肽(

B类

) circPLOD2a (

)circPLOD2a(

= 73) or (

（73）或（

) circPLOD2b (

)circPLOD2b(

= 54) compared with corresponding control lanes.

=54）与相应的控制车道进行比较。

F级

Potential proteins interacting with (

潜在的蛋白质与(

) circPLOD2a and (

)circPLOD2a和(

F级

) circPLOD2b were verified by RNA pull-down assays with corresponding antisense probes in hypoxic U87 cells. Relative integrated density normalized to β-actin was marked above each band.

)通过在缺氧U87细胞中用相应的反义探针进行RNA下拉测定来验证circPLOD2b。在每个条带上方标记了归一化为β-肌动蛋白的相对积分密度。

克

–

RIP assay confirms the interaction of circPLOD2a/b and linear

RIP分析证实了circPLOD2a/b与线性的相互作用

PLOD2

with HuR. CircPLOD2a/b and linear

与HuR。CircPLOD2a/b和线性

PLOD2

were detected by RT-PCR (

通过RT-PCR检测(

克

). The immunoprecipitation products of anti-HuR compared with IgG were treated with or without RNase R. Quantitative enrichment analysis of circPLOD2a (

)。与IgG相比，抗HuR的免疫沉淀产物用或不用RNase R处理。circPLOD2a的定量富集分析(

小时

), circPLOD2b (

)，circPLOD2b(

我

) and linear

)和线性

PLOD2

(

) were analyzed by qRT-PCR,

)通过qRT-PCR进行分析，

= 3.

= 3。

Cellular localization of circPLOD2b (red) and HuR (green) were examined by dual RNA-FISH and IF in U87 cells treated with normoxic and hypoxic conditions. Scale bar: 25 μm.

在常氧和低氧条件下处理的U87细胞中，通过双RNA-FISH和IF检查circPLOD2b（红色）和HuR（绿色）的细胞定位。比例尺：25μm。

Schematic diagram of wild type and various truncated HuR with Flag-tag. (M) The interaction of circPLOD2a/b with full-length or truncated HuR protein were detected by RIP assay in U87 cells. CircPLOD2a/b were detected by RT-PCR; full-length and truncated HuR proteins were detected by western blot using anti-Flag antibody.

野生型和带有Flag标签的各种截短的HuR的示意图。（M）通过RIP测定在U87细胞中检测circPLOD2a/b与全长或截短的HuR蛋白的相互作用。通过RT-PCR检测CircPLOD2a/b；使用抗Flag抗体通过蛋白质印迹检测全长和截短的HuR蛋白。

CircPLOD2a and circPLOD2b were immunoprecipitated by anti-Flag beads in U87 cells transfected with plasmids carrying HuR- wildtype (HuR-wt) or truncated forms (HuR-

在用携带HuR野生型（HuR wt）或截短型（HuR wt）的质粒转染的U87细胞中，通过抗Flag珠免疫沉淀CircPLOD2a和circPLOD2b-

△

RRM1/2/3) and detected by qRT-PCR,

，

= 3. Data are shown as mean ± SEM (error bars) and analyzed using Student’s

数据显示为平均SEM（误差线），并使用学生的

-test (

-测试(

小时

–

) and ANOVA (

)和方差分析(

Full size image

全尺寸图像

To probe the structural basis of the interactions between circPLOD2a/b and HuR, we constructed plasmids carrying Flag-tagged wild type HuR (HuR-wt) and its three truncated forms (HuR-

为了探讨circPLOD2a/b与HuR之间相互作用的结构基础，我们构建了携带Flag标签的野生型HuR（HuR wt）及其三种截短形式（HuR）的质粒-

△

RRM1/2/3) containing different functional domains (Fig.

RRM1/2/3）包含不同的功能域（图）。

4升

). RIP assay showed that RRM1 and RRM2 domains in HuR are essential for its interaction with circPLOD2a, while all three RRMs domains are involved in binding to circPLOD2b in hypoxic GBM cells (Fig.

)。RIP分析表明，HuR中的RRM1和RRM2结构域对于其与circPLOD2a的相互作用至关重要，而所有三个RRMs结构域都参与缺氧GBM细胞中与circPLOD2b的结合（图）。

4M, N

4米，北纬

and Supplementary Fig.

和补充图。

9天

). The potential binding regions of circPLOD2a/b were predicted using the catRAPID database (

)。使用catRAPID数据库预测了circPLOD2a/b的潜在结合区(

http://service.tartaglialab.com/page/catrapid_group

). The results indicate that the binding region of circPLOD2a to HuR is likely located between 26–80 nt. For circPLOD2b, multiple binding sites are predicted at 26–80 nt, 268–319 nt, and 418–644 nt (Supplementary Fig.

)。结果表明，circPLOD2a与HuR的结合区可能位于26-80 nt之间。对于circPLOD2b，预测多个结合位点位于26-80 nt，268-319 nt和418-644 nt（补充图。

3A, B

3A，B

Taken together, our results suggest that circPLOD2a/b directly interact with HuR, which might explain the regulatory role of circPLOD2a/b in the invasion and migration of GBM cells under hypoxia.

综上所述，我们的结果表明circPLOD2a/b直接与HuR相互作用，这可能解释了circPLOD2a/b在缺氧条件下GBM细胞侵袭和迁移中的调节作用。

CircPLOD2a/b promote cell invasion and migration by suppressing

CircPLOD2a/b通过抑制细胞侵袭和迁移

XIRP1

through interaction with HuR

通过与HuR的互动

The human RBP HuR is a conserved mRNA stability regulator

人RBP-HuR是一种保守的mRNA稳定性调节剂

. To identify the downstream molecules of circPLOD2a/b/HuR regulatory axis, we analyzed the differentially expressed genes (DEGs) by performing RNA-seq (GSE245636) in KD-circPLOD2a U87 cells compared with sh-Scramble, and identified 57 up-regulated together with 114 down-regulated genes ( | log2fold-change | ≥ 1 and .

为了鉴定circPLOD2a/b/HuR调控轴的下游分子，我们通过在KD-circPLOD2a U87细胞中进行RNA-seq（GSE245636）分析了差异表达基因（DEGs），与sh Scramble相比，鉴定了57个上调基因和114个下调基因（log2倍变化≥1和）。

< 0.05) (Fig.

<0.05）（图。

5A, B

5A、B

). We examined the expression of 15 up-regulated genes in both U87 KD- and OE-circPLOD2a by qRT-PCR, and identified 4 candidate genes which were up-regulated in KD-circPLOD2a cells and meanwhile down-regulated in OE-circPLOD2a cells (Fig.

)。我们通过qRT-PCR检测了U87 KD和OE-circPLOD2a中15个上调基因的表达，并鉴定了4个候选基因，这些基因在KD-circPLOD2a细胞中上调，同时在OE-circPLOD2a细胞中下调（图）。

5C, D

5C，D

; Supplementary Fig.

。

4A, B

4A，B

Fig. 5: CircPLOD2a/b promote GBM cell invasion and migration by suppressing

图5:CircPLOD2a/b通过抑制GBM细胞侵袭和迁移

XIRP1

through binding to HuR.

通过绑定到HuR。

Heatmap and (

热图和(

B类

) Volcano plot of DEGs in KD-circPLOD2a U87 cells compared to negative control cells under hypoxia. In total

)与缺氧条件下的阴性对照细胞相比，KD-circPLOD2a U87细胞中DEG的火山图。总计

= 57 up-regulated and

=57上调和

= 114 up-regulated genes were displayed.

显示了114个上调的基因。

C级

Expression of 4 up-regulated genes in RNA-seq data in (

RNA-seq数据中4个上调基因的表达(

C级

) KD-circPLOD2a and (

)KD-circPLOD2a和(

) OE-circPLOD2a U87 cells examined by qRT-PCR.

)通过qRT-PCR检查OE-circPLOD2a U87细胞。

Expression of these 4 genes in KD-HuR cells.

。

F级

克

Expression of

表达

XIRP1

in (

在(

F级

) KD-circPLOD2b and (

)KD-circPLOD2b和(

克

) OE-circPLOD2b cells.

OE-circPLOD2b细胞。

小时

我

Binding of (

绑定(

小时

) circPLOD2a and (I)

)circPLOD2a和（I）

XIRP1

to HuR were detected by RIP assay in U87 OE-circPLOD2a and control cells.

通过RIP测定在U87 OE-circPLOD2a和对照细胞中检测到HuR。

Binding of (

绑定(

) circPLOD2b and (

)(

)

XIRP1

to HuR were detected by RIP assay in U87 OE-circPLOD2b and control cells.

通过RIP测定在U87 OE-circPLOD2b和对照细胞中检测到HuR。

The expression (

表达式(

) mRNA and (

)mRNA和(

) protein level of

)蛋白质水平

XIRP1

were rescued by overexpression of HuR in OE-circPLOD2a and OE-circPLOD2b cells. Relative integrated density normalized to β-actin was marked above each band.

通过在OE-circPLOD2a和OE-circPLOD2b细胞中过表达HuR来挽救。在每个条带上方标记了归一化为β-肌动蛋白的相对积分密度。

OE-HuR U87 and LN229 cells were treated with actinomycin D (5 μg/ml). The relative expression of

用放线菌素D（5μg/ml）处理OE HuR U87和LN229细胞。的相对表达式

XIRP1

was detected by qRT-PCR at different time points post actinomycin D treatment.

在放线菌素D处理后的不同时间点通过qRT-PCR检测。

Transwell invasion assays illustrate that knockdown of

Transwell入侵分析表明

XIRP1

in (

在(

) KD-circPLOD2a or (

)KD-circPLOD2a或(

) KD-circPLOD2b U87 cells could rescue the inhibition of cell invasion and migration induced by knockdown of these two circRNAs under hypoxia. scale bar: 100 μm. (Q and R) Wound healing assays illustrate that knockdown of

)KD-circPLOD2b U87细胞可以挽救缺氧条件下敲低这两种circRNA诱导的细胞侵袭和迁移的抑制。比例尺：100微米。（Q和R）伤口愈合试验表明

XIRP1

in (

在(

) KD-circPLOD2a or (

)KD-circPLOD2a或(

) KD-circPLOD2b U87 cells could rescue the inhibition of cell migration ability under hypoxia. Scale bar: 250 μm.

)KD-circPLOD2b U87细胞可以挽救缺氧条件下细胞迁移能力的抑制。比例尺：250微米。

= 3 replicates unless otherwise noted. Data are shown as mean ± SEM (error bars) and analyzed using ANOVA. Scr: Scramble.

除非另有说明，否则重复3次。数据显示为平均值±SEM（误差线），并使用ANOVA进行分析。Scr：加扰。

Full size image

全尺寸图像

We constructed KD-

我们构建了KD-

HuR

cell line in U87 (Supplementary Figs.

U87细胞系（补充图）。

4C级

), and further inspected the mRNA expression levels of candidate genes by qRT-PCR.

)，并通过qRT-PCR进一步检查候选基因的mRNA表达水平。

XIRP1

and

和

ANGPTL6

were found to be down-regulated, while

被发现下调，而

GLRA2

was up-regulated in KD-

KD上调-

HuR

cells (Fig.

细胞（图）。

5E级

). Consistent with the regulatory effects of circPLOD2a on

)。与circPLOD2a对

XIRP1

expression,

表达式，

XIRP1

was also up-regulated in KD-circPLOD2b cells and down-regulated in OE-circPLOD2b cells (Fig.

在KD-circPLOD2b细胞中也上调，在OE-circPLOD2b细胞中下调（图）。

5F, G

5F，G

). Previous studies have implied

)。先前的研究暗示

XIRP1

as a potential tumor suppressor

作为潜在的肿瘤抑制因子

, which is in tune with the promotion of GBM cell invasion and migration by circPLOD2a/b here. Thus, we chose

，这与circPLOD2a/b促进GBM细胞侵袭和迁移相一致。因此，我们选择了

XIRP1

for further investigation.

以便进一步调查。

RIP assay was conducted in OE-circPLOD2a or OE-circPLOD2b U87 cells with anti-HuR antibody (Fig.

用抗HuR抗体在OE-circPLOD2a或OE-circPLOD2b U87细胞中进行RIP测定（图）。

5H, J

5H，J

). There was an appreciable enrichment of

)。有一个可观的丰富

XIRP1

by anti-HuR antibody pulldown compared with IgG control, indicating that

与IgG对照相比，通过抗HuR抗体下拉，表明

XIRP1

can bind to HuR.

可以绑定到HuR。

Hypoxia decreased

缺氧减少

XIRP1

mRNA enrichment in the RIP assay (Supplementary Fig.

RIP分析中的mRNA富集（Supplementary Fig.）。

4D级

), likely driven by the hypoxia-induced upregulation of circPLOD2a/b. Overexpression of circPLOD2a (Fig.

)，可能是由缺氧诱导的circPLOD2a/b上调驱动的。circPLOD2a的过表达（图）。

5I号

) or circPLOD2b (Fig.

)或circPLOD2b（图）。

5J型

) reduced XIRP1 mRNA enrichment in the RIP assay. Conversely,

)RIP分析中XIRP1 mRNA的富集减少。相反，

XIRP1

immunoprecipitation increased following the knockdown of circPLOD2a/b in U87 cells (Supplementary Fig.

在U87细胞中敲除circPLOD2a/b后，免疫沉淀增加（Supplementary Fig.）。

E–H

). These findings suggest that circPLOD2a/b may function as protein sponges, binding to HuR to inhibit XIRP1.

)。这些发现表明circPLOD2a/b可能起蛋白质海绵的作用，与HuR结合以抑制XIRP1。

To verify that circPLOD2a/b regulate the expression of

验证circPLOD2a/b调节

XIRP1

through HuR, we overexpressed HuR in U87 OE-circPLOD2a or OE-circPLOD2b cells. The results showed that overexpression of HuR could effectively rescue the downregulation of

通过HuR，我们在U87 OE-circPLOD2a或OE-circPLOD2b细胞中过表达HuR。结果表明，HuR的过表达可以有效地挽救HuR的下调

XIRP1

induced by circPLOD2a/b (Fig.

由circPLOD2a/b诱导（图）。

5L, M

5升，米

and Supplementary Fig.

和补充图。

We examined the stability of

我们检查了

XIRP1

mRNA in OE-HuR cells followed by actinomycin D treatment, and found that the stability of

放线菌素D处理后，OE-HuR细胞中的mRNA表达，发现其稳定性

XIRP1

was enhanced in U87 cells with HuR overexpression, compared with control cells (Fig.

与对照细胞相比，HuR过表达的U87细胞增强（图）。

5牛

). Similarly, knockdown of circPLOD2a and circPLOD2b improved the half-life of

)。同样，击倒circPLOD2a和circPLOD2b可以提高半衰期

XIRP1

mRNA compared to control cells, indicated an inhibited degradation of

与对照细胞相比，mRNA的降解受到抑制

XIRP1

caused by Actinomycin D treatment in U87 and LN229 under hypoxia. (Supplementary Fig.

由放线菌素D在缺氧条件下在U87和LN229中处理引起。（补充图。

5A–D

). Therefore, HuR may stabilize

)。因此，HuR可能会稳定

XIRP1

, which could be disrupted by competitive binding of circPLOD2a/b and consequently lead to the degradation of

，这可能会被circPLOD2a/b的竞争性结合所破坏，从而导致

XIRP1

The regulatory role of HuR on the translation of

HuR对翻译的调节作用

XIRP1

was investigated by sucrose gradient ribosome separation assay in U87 KD-HuR and control cells. The knockdown of HuR led to a significant decrease in

通过蔗糖梯度核糖体分离测定法在U87 KD HuR和对照细胞中进行了研究。HuR的击倒导致

XIRP1

mRNA within polysome association, indicating HuR enhance the translational efficiency of

多核糖体关联中的mRNA，表明HuR增强了

XIRP1

(Supplementary Fig.

（补充图。

5E–G

We further explored whether the effect of circPLOD2a/b on GBM cell invasion and migration is dependent on the regulation of

我们进一步探讨了circPLOD2a/b对GBM细胞侵袭和迁移的影响是否依赖于

XIRP1

. Knockdown of

.击倒

XIRP1

could effectively attenuate the inhibition of cell invasion and migration induced by knockdown of circPLOD2a/b in hypoxic U87 cells in transwell assay (Fig.

在transwell试验中，可以有效地减弱缺氧U87细胞中circPLOD2a/b敲低诱导的细胞侵袭和迁移的抑制（图）。

5O, P

5O，P

). Wound-healing assay further confirmed this hypothesis as knockdown of

)。伤口愈合试验进一步证实了这一假设

XIRP1

in KD-circPLOD2a or KD-circPLOD2b U87 cells could rescue the inhibition of cell migration ability under hypoxia (Fig.

在KD-circPLOD2a或KD-circPLOD2b中，U87细胞可以挽救缺氧条件下细胞迁移能力的抑制（图）。

5Q, R

5Q，R

Therefore, we demonstrate that circPLOD2a/b suppress

因此，我们证明circPLOD2a/b抑制

XIRP1

through competitively binding to HuR to interrupt the interaction of HuR and

通过与HuR竞争性结合来中断HuR与

XIRP1

mRNA, thus promoting invasion and migration of GBM cells under hypoxia.

mRNA，从而促进缺氧条件下GBM细胞的侵袭和迁移。

CircPLOD2a/b promote the progression of GBM xenografts in mice by suppressing

CircPLOD2a/b通过抑制小鼠GBM异种移植物的进展

XIRP1

We take a close look at the function of circPLOD2a/b-HuR-

我们仔细研究了circPLOD2a/b-HuR的功能-

XIRP1

regulatory axis in GBM progression in vivo using cell line-derived xenograft (CDX) model in mice. Four-week-old BALB/c nude mice were randomly allocated into five groups (control-luc, KD-circPLOD2a-luc, KD-circPLOD2a/KD-

在小鼠中使用细胞系衍生的异种移植物（CDX）模型在体内GBM进展中的调节轴。将4周龄BALB/c裸鼠随机分为5组（对照组luc，KD-circPLOD2a-luc，KD-circPLOD2a/KD-

XIRP1

-luc, KD-circPLOD2b-luc, KD-circPLOD2b/KD-

-luc，KD-circPLOD2b-luc，KD-circPLOD2 b/KD-

XIRP1

-luc). Mice from each group were intracranially transplanted with corresponding genetically modified U87-luc cells (Fig.

-卢克）。每组小鼠颅内移植相应的转基因U87-luc细胞（图）。

). Decreased bioluminescence was detected in KD-circPLOD2a and KD-circPLOD2b groups compared with the control group, indicating that knockdown of circPLOD2a/b inhibits the expansion of GBM cells in vivo. This phenotype could be rescued by

)。与对照组相比，KD-circPLOD2a和KD-circPLOD2b组的生物发光降低，表明circPLOD2a/b的敲低抑制了体内GBM细胞的扩增。这种表型可以通过

XIRP1

knockdown as disclosed by the increased bioluminescence in KD-circPLOD2a/KD-

KD-circPLOD2a/KD中生物发光增加所揭示的击倒-

XIRP1

and KD-circPLOD2b/KD-

KD-crcPLOD2b/KD-

XIRP1

groups compared with KD-circPLOD2a and KD-circPLOD2b groups, respectively (Fig.

组分别与KD-circPLOD2a和KD-circPLOD2b组比较（图）。

6A, B

). Body weights of tumor-bearing mice from all groups were stable during trial period (Supplementary Fig.

)。来自所有组的荷瘤小鼠的体重在试验期间是稳定的（Supplementary Fig.）。

Fig. 6: CircPLOD2a/b promote invasion and migration of GBM cells by suppressing

图6:CircPLOD2a/b通过抑制GBM细胞的侵袭和迁移

XIRP1

in vivo.

。

B类

(

) Representative bioluminescence images and (

)代表性的生物发光图像和(

B类

) tumor growth curve of tumor-bearing mice intracranially transplanted with corresponding genetically modified U87 cells labeled with luciferase (

)荧光素酶标记的相应转基因U87细胞颅内移植荷瘤小鼠的肿瘤生长曲线(

= 5–6/ per group).

每组5-6）。

C级

Relative expression of

相对表达式

XIRP1

in CDX tumors,

在CDX肿瘤中，

= 3.

= 3。

Representative images of IHC staining of Vimentin, E-cadherin and N-cadherin in CDX tumors. Scale bar: 100μm.

CDX肿瘤中波形蛋白，E-钙粘蛋白和N-钙粘蛋白的IHC染色的代表性图像。比例尺：100微米。

F级

Relative protein level of Vimentin, E-cadherin, N-cadherin, HuR and XIRP1 in different genetically-modified (

不同基因修饰的波形蛋白，E-钙粘蛋白，N-钙粘蛋白，HuR和XIRP1的相对蛋白水平(

) U87 cells in vitro and in (

)U87细胞的体外和体内(

F级

) CDX tumors in vivo. Relative integrated density normalized to β-actin was marked above each band. Data are shown as mean ± SEM (error bars) and analyzed ANOVA. Scr: Scramble.

)体内CDX肿瘤。在每个条带上方标记了归一化为β-肌动蛋白的相对积分密度。数据显示为平均值±SEM（误差线）并分析ANOVA。Scr：加扰。

Full size image

全尺寸图像

Mice were then sacrificed at 52 days post transplantation, and the tumor tissues were collected for further analysis. Epithelial-mesenchymal transition (EMT) is a process that epithelial cells lose their cell-cell adhesion and obtain mesenchymal features, which is critical for tumor metastasis. Thus, we examined the expression of EMT markers in the CDX tumors by western-blot and IHC staining.

然后在移植后52天处死小鼠，收集肿瘤组织用于进一步分析。上皮-间质转化（EMT）是上皮细胞失去细胞间粘附并获得间充质特征的过程，这对于肿瘤转移至关重要。因此，我们通过蛋白质印迹和IHC染色检查了CDX肿瘤中EMT标志物的表达。

The results showed that tumors derived from KD-circPLOD2a/b group had higher level of E-cadherin and lower level of Vimentin and N-cadherin (Fig. .

结果表明，KD-circPLOD2a/b组肿瘤的E-钙粘蛋白水平较高，波形蛋白和N-钙粘蛋白水平较低（图）。

6天

), suggesting that knockdown of these two circRNAs could inhibit the EMT process of GBM cells in vivo. Consistent with the results of CDX tumor expansion and the in vitro experiments, the effects of KD-circPLOD2a/b on EMT marker expression could be effectively inhibited by knockdown of

)，表明这两种circRNA的敲低可以抑制体内GBM细胞的EMT过程。与CDX肿瘤扩增和体外实验的结果一致，KD-circPLOD2a/b对EMT标志物表达的影响可以通过敲除

XIRP1

. Besides, knockdown of circPLOD2a/b had no effect on the protein level of HuR (Fig.

此外，circPLOD2a/b的敲低对HuR的蛋白质水平没有影响（图）。

6E, F

6E，F

, Supplementary Fig.

，补充图。

11A, B

11A、B

Taken together, our results demonstrate that circPLOD2a/b inhibit the expansion of GBM xenografts by suppressing

综上所述，我们的结果表明circPLOD2a/b通过抑制GBM异种移植物的扩增来抑制GBM异种移植物的扩增

XIRP1

in vivo.

。

CircPLOD2a/b are highly expressed in GBM tissues and XIRP1 is negatively correlated with glioma grade and prognosis

CircPLOD2a/b在GBM组织中高表达，XIRP1与胶质瘤分级和预后呈负相关

To clarify the expression and clinical significance of circPLOD2a/b, qRT-PCR was conducted to detect the expression of circPLOD2a/b and

为了阐明circPLOD2a/b的表达及其临床意义，进行了qRT-PCR检测circPLOD2a/b和

XIRP1

in 19 clinical specimens containing grade II-III (

在19个II-III级临床标本中(

= 10) and IV (

(

= 9) glioma. The results showed a remarkable enrichment of circPLOD2a/b in GBM compared with lower grade glioma (Fig.

9）神经胶质瘤。结果显示，与低级别胶质瘤相比，GBM中circPLOD2a/b显着富集（图）。

7A, B

7A，B

), and the expression of downstream gene

)，以及下游基因的表达

XIRP1

was suppressed in circPLOD2a/b-high group (Fig.

在circPLOD2a/b高组中被抑制（图）。

7C, D

7C、D

). These findings suggest that circPLOD2a/b are highly expressed in GBM and negatively correlated with the expression of XIRP1.

)。这些发现表明circPLOD2a/b在GBM中高度表达，并与XIRP1的表达呈负相关。

Fig. 7: CircPLOD2a/b are highly expressed in GBM tissues and its downstream gene

图7:CircPLOD2a/b在GBM组织及其下游基因中高度表达

XIRP1

is correlated with glioma grade and prognosis.

与胶质瘤分级和预后相关。

B类

Relative expression of (

相对表达式(

) circPLOD2a and (

)circPLOD2a和(

B类

) circPLOD2b were examined by qRT-PCR in clinical samples from patients with grade II-III (

)通过qRT-PCR检测II-III级患者临床样本中的circPLOD2b(

= 10) and grade IV (

=10）和IV级(

= 9) glioma.

（9）glioma。

C级

Relative expression of

相对表达式

XIRP1

in tissues from circPLOD2a-high and -low groups. The GBM tissues were divided into circPLOD2a-high (

在来自circPLOD2a高和低组的组织中。GBM组织分为circPLOD2a高(

= 10) and -low (

=10）和-低(

= 9) groups according to the expression of circPLOD2a.

= 9）根据circPLOD2a的表达分组。

Relative expression of

相对表达式

XIRP1

in tissues from circPLOD2b-high and -low groups. The GBM tissues were divided into circPLOD2a-high (

在来自circPLOD2b高和低组的组织中。GBM组织分为circPLOD2a高(

= 10) and -low (

=10）和-低(

= 9) groups according to the expression of circPLOD2b.

= 9）根据circPLOD2b的表达分组。

Relative expression of

相对表达式

XIRP1

in GBM and non-tumor brain tissues were analyzed from Rembrandt database.

在GBM和非肿瘤脑组织中，从伦勃朗数据库中进行了分析。

F级

Representative immunohistochemistry images of XIRP1 in a tissue microarray containing grade I (

包含I级的组织微阵列中XIRP1的代表性免疫组织化学图像(

= 3), II (

= 3），ii（

= 50), III (

==50），III(

= 24) and IV (

24）和IV(

= 94) glioma. Scale bar: 100 μm.

94）神经胶质瘤。比例尺：100微米。

克

小时

Expression levels of XIRP1 in tissues from patients with different grade glioma were evaluated by (

通过以下方法评估XIRP1在不同级别胶质瘤患者组织中的表达水平(

克

) the percentage of cells with strong/moderate/weak intensity of XIRP1 staining and (

)XIRP1染色强度强/中/弱的细胞百分比(

小时

) histochemistry score (H-score) of each tissue on this microarray.

)该微阵列上每个组织的组织化学评分（H评分）。

我

Kaplan–Meier survival curves of glioma patients in XIRP1-high and low groups. Glioma patients were categorized into XIRP1-high and -low group by the expression level of XIRP1 based on H-score.

XIRP1高组和低组胶质瘤患者的Kaplan-Meier生存曲线。根据H评分，根据XIRP1的表达水平将胶质瘤患者分为XIRP1高组和低组。

Schematic illustration of HIF1α/circPLOD2a/b/HuR/XIRP1 axis. HIF1α induces the up-regulation of linear

HIF1α/circPLOD2a/b/HuR/XIRP1轴的示意图。HIF1α诱导线性上调

PLOD2

, circPLOD2a and circPLOD2b under hypoxia. CircPLOD2a/b suppress

，缺氧条件下的circPLOD2a和circPLOD2b。CircPLOD2a/b抑制

XIRP1

by competitively binding to HuR and enhance GBM cell aggressiveness. Data are shown as mean ± SEM (error bars) and analyzed using Student’s

通过竞争性结合HuR并增强GBM细胞的侵袭性。数据显示为平均SEM（误差线），并使用学生的

-test (

-测试(

–

), ANOVA (

)，方差分析(

小时

) and Log-rank test (

)和对数秩检验(

我

Full size image

全尺寸图像

We further explored the expression and clinical significance of

我们进一步探讨了

XIRP1

. The expression of

.表达

XIRP1

was lower in GBM than non-tumor tissues according the data from Rembrandt database (Fig.

根据伦勃朗数据库的数据，GBM低于非肿瘤组织（图）。

7E型

)

. Immunohistochemical analysis of XIRP1 was performed in tissue microarray which contains grade I (

.在含有I级的组织微阵列中进行XIRP1的免疫组织化学分析(

= 3), II (

= 3），ii（

= 50), III (

= 50），三（

= 24) and IV (

24）和IV(

= 94) glioma patient tissues. Histochemistry score (H score) was calculated to evaluate the expression level of XIRP1 in each tissue. The results showed that XIRP1 decreased with the increase of glioma grade, and the expression level of XIRP1 was significantly lower in grade IV than grade II (Fig. .

94）胶质瘤患者组织。计算组织化学评分（H评分）以评估每个组织中XIRP1的表达水平。结果显示，XIRP1随着胶质瘤分级的增加而降低，IV级XIRP1的表达水平显着低于II级（图）。

7F–H

). Survival analysis of IHC data of tissue microarray indicated that patients with low level of XIRP1 had worse overall survival than those with high XIRP1 level (Fig.

)。组织微阵列IHC数据的生存分析表明，XIRP1水平低的患者总生存率低于XIRP1水平高的患者（图）。

7I个

As a summary, circPLOD2a/b are highly expressed in GBM, while its downstream gene

总之，circPLOD2a/b在GBM中高度表达，而其下游基因

XIRP1

was suppressed and low expression of

被抑制并且低表达

XIRP1

is correlated with higher glioma grade and worse prognosis (Fig.

与较高的胶质瘤分级和较差的预后相关（图）。

7J型

Discussion

讨论

To gain essential elements such as oxygen and glucose, tumors tend to metastasize under hypoxic microenvironment. A growing number of circRNAs have been implicated in the regulation of cell proliferation, transformation and metastasis of malignant tumors

为了获得氧气和葡萄糖等必需元素，肿瘤倾向于在低氧微环境下转移。越来越多的circRNA参与了恶性肿瘤细胞增殖、转化和转移的调控

. Brain tumors such as GBM usually exist in a low-oxygen environment as human brain has lower oxygen concentration than other organs. However, the role of circRNAs responding to hypoxia in brain tumors is still unclear. In order to disclose the circRNAs that are involved in hypoxic responses in GBM, we screened circRNAs induced by hypoxia in U87 cells by RNA sequencing.

。GBM等脑肿瘤通常存在于低氧环境中，因为人脑的氧浓度低于其他器官。然而，circRNA在脑肿瘤中对缺氧反应的作用仍不清楚。为了揭示参与GBM缺氧反应的circRNA，我们通过RNA测序筛选了U87细胞缺氧诱导的circRNA。

Here, we identified two hypoxia-induced circRNAs, which were significantly up-regulated by HIF1α and could promote GBM cell invasion and migration under hypoxia both in vitro and in vivo. These two circRNAs, circPLOD2a and circPLOD2b, were derived from the same parental gene PLOD2. Mechanically, circPLOD2a/b directly bind to HuR and inhibit its interaction with .

在这里，我们鉴定了两种缺氧诱导的circRNA，它们被HIF1α显着上调，并且可以在体外和体内缺氧条件下促进GBM细胞的侵袭和迁移。这两个circRNA，circPLOD2a和circPLOD2b，来自同一亲本基因PLOD2。机械地，circPLOD2a/b直接与HuR结合并抑制其与HuR的相互作用。

XIRP1

mRNA, leading to downregulation of

mRNA，导致下调

XIRP1

. Therefore, our findings have revealed the oncogenic role of hypoxia-induced circPLOD2a/b in GBM cells under hypoxia, and uncovered the underlying mechanisms.

因此，我们的研究结果揭示了缺氧诱导的circPLOD2a/b在缺氧条件下GBM细胞中的致癌作用，并揭示了潜在的机制。

Recent studied revealed that circRNAs have various biological functions, including miRNA sponges

最近的研究表明，circRNA具有多种生物学功能，包括miRNA海绵

, binding with protein partners

，与蛋白质伴侣结合

, modulating the expression of parental genes

，调节亲本基因的表达

and serving as protein-coding sequences for short peptides

并作为短肽的蛋白质编码序列

. To figure out whether circPLOD2a/b regulate the aggressiveness of GBM through other mechanisms besides competing with

。了解circPLOD2a/b是否通过除与竞争之外的其他机制调节GBM的攻击性

XIRP1

mRNA to interact with HuR, we analyzed the sequences of circPLOD2a/b in circRNADb database (

为了与HuR相互作用，我们分析了circRNADb数据库中circPLOD2a/b的序列(

http://reprod.njmu.edu.cn/cgi-bin/circrnadb/circRNADb.php

)

, which showed that the possibilities of protein coding for these two circRNAs are relatively low (

，这表明蛋白质编码这两个circRNA的可能性相对较低(

< 1.6). Besides, circRNAs act as miRNA sponge are widely reported, in which AGO2 is essential for miRNA recruitment

此外，circRNA作为miRNA海绵被广泛报道，其中AGO2对于miRNA的募集至关重要

. In our results of pull-down mass spectrometry, AGO2 was not detected, which implies that circPLOD2a/b probably does not act as miRNA sponges.

在我们的下拉质谱结果中，未检测到AGO2，这意味着circPLOD2a/b可能不充当miRNA海绵。

The proliferation and metastasis are hallmarks of cancer progression, which are modulated by various signaling pathways such as PI3K/AKT/mTOR and TGF-beta

增殖和转移是癌症进展的标志，受PI3K/AKT/mTOR和TGF-β等多种信号通路的调节

. CircRNAs participate in the regulation of tumor cell metastasis. It was reported that circRHOBTB3 and circEXOC6B repress tumor cell metastasis by decreasing epithelial-mesenchymal transition (EMT) in colorectal and prostate cancer respectively

circRNA参与肿瘤细胞转移的调节。据报道，circRHOBTB3和circEXOC6B分别通过降低结直肠癌和前列腺癌的上皮-间质转化（EMT）来抑制肿瘤细胞转移

. Under hypoxic microenvironment, tumor cells tend to restrain the cell proliferation and develop more aggressive phenotype

在低氧微环境下，肿瘤细胞倾向于抑制细胞增殖并发展出更具侵袭性的表型

. In this process, HIF1α plays an important role to enhance EMT, leading to cell migration and invasion

在此过程中，HIF1α在增强EMT中起重要作用，导致细胞迁移和侵袭

. Consistent with the above theory of hypoxia inducing metastasis, circPLOD2a/b promote cell invasion and migration while had no significant influence on the proliferation of GBM cells under hypoxia.

与上述缺氧诱导转移的理论一致，circPLOD2a/b促进细胞侵袭和迁移，而对缺氧条件下GBM细胞的增殖没有显着影响。

In this study, we observed that the circular isoforms of PLOD2 (circPLOD2a and circPLOD2b) were significantly upregulated following HIF1α overexpression and correspondingly downregulated upon HIF1α knockdown in U87 cells. Additionally, our luciferase reporter assay confirmed that HIF1α significantly enhances .

在这项研究中，我们观察到PLOD2的环状同种型（circPLOD2a和circPLOD2b）在HIF1α过表达后显着上调，并且在U87细胞中HIF1α敲低后相应下调。。

PLOD2

promoter activity, providing strong evidence that HIF1α transcriptionally regulates the expression of circPLOD2a/b under hypoxic conditions. However, the exact mechanism linking HIF1α-mediated transcription of PLOD2 and its circular isoform generation, such as whether the circularization occurs spontaneously or concurrently with transcription, remains to be fully elucidated.

启动子活性，提供了强有力的证据表明HIF1α在低氧条件下转录调节circPLOD2a/b的表达。然而，将HIF1α介导的PLOD2转录与其环状同工型产生联系起来的确切机制，例如环化是自发发生还是与转录同时发生，仍有待完全阐明。

Investigating the alternative splicing or back-splicing events associated with circPLOD2 formation following HIF1α expression modulation is one of the key directions for future studies..

研究HIF1α表达调节后与circPLOD2形成相关的选择性剪接或反向剪接事件是未来研究的关键方向之一。。

It was reported that

据报道

PLOD2

, the parental gene of circPLOD2a/b, promotes GBM cell proliferation and invasion under hypoxia by regulating downstream mRNA such as

，circPLOD2a/b的亲本基因，通过调节下游mRNA（例如

MT1-MMP

CD44

CD44细胞

CD99

, and

，以及

MMP2

基质金属蛋白酶2

. Previous studies reported that the expression of parental genes could be modulated by corresponding circRNAs in some cases. For example, CircSEP3 binds to its cognate DNA locus and pause the transcriptional of

先前的研究报道，在某些情况下，亲本基因的表达可以被相应的circRNA调节。例如，CircSEP3与其同源DNA基因座结合并暂停转录

SEP3

9月3日

by forming R-loops

通过形成R环

. Nuclei-localized circRNA ci-ankrd52 suppresses host gene

细胞核定位的circRNA ci-ankrd52抑制宿主基因

ANKRD52

through RNase H1

. Thus, it is possible that circPLOD2a/b could also promote cell metastasis by regulating its parental gene

因此，circPLOD2a/b也可能通过调节其亲本基因来促进细胞转移

PLOD2

. However, both the mRNA and protein levels of PLOD2 were not altered when circPLOD2a/b was knocked down or overexpressed (Supplementary Figs.

然而，当circPLOD2a/b被敲低或过表达时，PLOD2的mRNA和蛋白质水平都没有改变（补充图）。

), which indicates that circPLOD2a/b may not modulate the expression of its parental gene. Besides, we found that overexpression of circPLOD2a or circPLOD2b have no effect on the expression levels of each other (Supplementary Fig.

)，这表明circPLOD2a/b可能不会调节其亲本基因的表达。此外，我们发现circPLOD2a或circPLOD2b的过表达对彼此的表达水平没有影响（Supplementary Fig.）。

). Considering the fact that circPLOD2a and circPLOD2b directly bind to the same RNA binding protein HuR, the potential synergistic effects of circPLOD2a/b could be further explored. Taken together, our model is that both the linear transcript of

)。考虑到circPLOD2a和circPLOD2b直接结合相同的RNA结合蛋白HuR的事实，可以进一步探索circPLOD2a/b的潜在协同作用。综上所述，我们的模型是

PLOD2

, and its circular forms, circPLOD2a/b are up-regulated by HIF1α in GBM cells under hypoxic microenvironment to promote invasion and migration, while their expression are not affected by each other.

，及其环状形式circPLOD2a/b在低氧微环境下被GBM细胞中的HIF1α上调以促进侵袭和迁移，而它们的表达不受彼此影响。

HuR, a vital RNA binding protein in tumor progression and metastasis, has post-transcriptional influence on target RNAs

HuR是肿瘤进展和转移中重要的RNA结合蛋白，对靶RNA具有转录后影响

. It was reported that HuR promotes tumor invasion by interacting with the 3’ UTR and stabilizing coding mRNA such as

据报道，HuR通过与3'UTR相互作用并稳定编码mRNA（例如

snail

and

和

MMP9

MMP9公司

. Here, we identified HuR as the protein partner of circPLOD2a/b in the responses to hypoxia, but its expression is not regulated by circPLOD2a/b (Fig.

在这里，我们将HuR鉴定为circPLOD2a/b在缺氧反应中的蛋白质伴侣，但其表达不受circPLOD2a/b的调节（图）。

6E, F

6E，F

). Our results showed that HuR could positively regulate the expression of

)。

XIRP1

through directly binding with the mRNA of this gene. Therefore, HuR may stabilize XIRP1 mRNA, which is disrupted by competitive binding by circPLOD2a/b and consequently lead to the degradation of

通过直接与该基因的mRNA结合。因此，HuR可以稳定XIRP1 mRNA，XIRP1 mRNA被circPLOD2a/b的竞争性结合破坏，从而导致XIRP1 mRNA的降解

XIRP1

mRNA. Previous studies have shown that HuR plays an oncogenic role under hypoxic conditions by regulating gene expression at the post-transcriptional level

mRNA。先前的研究表明，HuR通过在转录后水平调节基因表达，在低氧条件下发挥致癌作用

. However, as a multi-target regulator, HuR is involved in the post-transcriptional regulation of various cancer-associated genes, with some promoting GBM aggressiveness

然而，作为一种多靶点调节因子，HuR参与了各种癌症相关基因的转录后调控，其中一些促进了GBM的侵袭性

and others inhibiting its progression

以及其他抑制其进展的因素

. Given the complex cellular functions of HuR, targeting the circPLOD2a/b–HuR–XIRP1 axis for GBM treatment should focus on inhibiting circPLOD2a/b expression or disrupting the interaction between XIRP1 and HuR, rather than directly downregulating HuR, to minimize potential side effects from other downstream targets..

鉴于HuR的复杂细胞功能，针对GBM治疗的circPLOD2a/b–HuR–XIRP1轴应侧重于抑制circPLOD2a/b表达或破坏XIRP1与HuR之间的相互作用，而不是直接下调HuR，以最大程度地减少其他下游靶标的潜在副作用。。

To summarize, our study identifies two hypoxia-response circRNAs, circPLOD2a and circPLOD2b, in GBM cell lines, which could be induced by HIF1α and directly interact with HuR to inhibit the mRNA level of

XIRP1

, thus promoting the aggressiveness of GBM cells both in vitro and in vivo. As a result, our study provides therapeutic targets for GBM.

，从而在体外和体内促进GBM细胞的侵袭性。因此，我们的研究为GBM提供了治疗靶点。

Methods

方法

Cell culture

细胞培养

Human GBM cell lines (DBTRG, LN229, T98G, U251 and U87) and HEK 293 T were purchased from American Type Culture Collection (ATCC). All cells were maintained in a humidified incubator at 37 °C with 5% CO

人GBM细胞系（DBTRG，LN229，T98G，U251和U87）和HEK 293T购自美国典型培养物保藏中心（ATCC）。所有细胞均保存在37°C、5%CO的潮湿培养箱中

and cultured in high glucose DMEM medium, supplemented with 10% fetal bovine serum, 100 mg/mL streptomycin and 100 U/mL penicillin. For the induction of hypoxia, cells were cultured in a tri-gas chamber (GeneScience E500, USA) with 1% O

并在补充有10%胎牛血清的高糖DMEM培养基中培养，100 mg/mL链霉素和100 U/mL青霉素。为了诱导缺氧，将细胞在含有1%O的三气室（GeneScience E500，USA）中培养

, 94% N

，94%氮

and 5% CO

和5%的CO

for 24 h if not indicated.

如果没有指示，则持续24小时。

RNA isolation, RNA sequencing, RNase R treatment and qRT-PCR

RNA分离，RNA测序，RNase R处理和qRT-PCR

Total RNA was extracted using TRIZOL reagent (Solarbio, China) and quantified by Nanodrop2000 spectrophotometer (Thermo Fisher Scientific, USA). RNA-seq was conducted using Illumina Hiseq by Novogene Co., Ltd (China). For RNase R treatment, 1ug of total RNA was treated with 0.05uL RNase R (Lucigen Middlesex, UK) for 10 min at 37 °C.

使用TRIZOL试剂（Solarbio，China）提取总RNA，并通过Nanodrop2000分光光度计（Thermo Fisher Scientific，USA）定量。RNA-seq由Novogene Co.，Ltd（中国）使用Illumina Hiseq进行。对于RNase R处理，将1ug总RNA用0.05uL RNase R（Lucigen Middlesex，UK）在37℃处理10分钟。

For qRT-PCR, the total RNA was synthesized into cDNA with PrimeScript RT Reagent Kit (Takara, China) in accordance with the manufacturer’s protocols. The cDNA was amplified with 2x M5 Ultra SYBR Mixture (Mei5bio, China) on a Bio-Rad CFX96 system (Bio-Rad, USA). The expression of circRNAs and mRNAs were analyzed by 2.

对于qRT-PCR，根据制造商的方案，使用PrimeScript RT试剂盒（Takara，China）将总RNA合成为cDNA。在Bio-Rad CFX96系统（Bio-Rad，USA）上用2x M5 Ultra SYBR混合物（Mei5bio，China）扩增cDNA。通过2分析circRNA和mRNA的表达。

–ΔΔCT

- ΔΔCT

, and normalized against β-actin. The primers were purchased from GeneCreate (China), and the sequences are detailed in Supplementary Table

，并针对β-肌动蛋白进行了标准化。引物购自GeneCreate（中国），序列详见补充表

Plasmids and cell transfection

质粒和细胞转染

For overexpression of circRNAs (OE-circPLOD2a/b), linear sequences of circPLOD2a/b were synthesized by GeneCreate (Wuhan, China) and subcloned into pLCDH-ciR vector. For HIF1α overexpression (OE-HIF1α), HIF1α coding sequence was obtained from HA-HIF1α-pcDNA3, which was purchased from Addgene (plasmid # 18949), and subcloned into pLenti-IRES-puro.

为了过表达circRNA（OE-circPLOD2a/b），由GeneCreate（中国武汉）合成了circPLOD2a/b的线性序列，并将其亚克隆到pLCDH-ciR载体中。对于HIF1α过表达（OE-HIF1α），从HA-HIF1α-pcDNA3获得HIF1α编码序列，其购自Addgene（质粒#18949），并亚克隆到pLenti-IRES-puro中。

3xFlag-tagged HuR protein was synthesized by GeneCreate (Wuhan, China) and subcloned into pLVX-IRES-neo vector. 3xFlag-tagged HuR RRM domain-truncated plasmids were amplified by overlapping PCR from pLVX-3xFlag-HuR-neo plasmid and cloned into pLVX-IRES-neo vector..

3xFlag标记的HuR蛋白由GeneCreate（中国武汉）合成，并亚克隆到pLVX-IRES-neo载体中。。。

Short-harpin RNA (shRNA) was used to knockdown gene expression. For knockdown of circRNA, shRNA targeting sites were designed at circinteractome (

短harpin RNA（shRNA）用于敲低基因表达。(

https://circinteractome.nia.nih.gov/

). For knockdown of HIF1α, HuR and XIRP1, shRNAs were designed at sigma Aldrich analyses (

)。为了敲除HIF1α，HuR和XIRP1，在sigma-Aldrich分析中设计了shRNA(

https://www.sigmaaldrich.cn/CN/zh/semi-configurators/shrna?activeLink=product

Search). Corresponding oligonucleotides targeting circPLOD2a/b and HIF1α were annealed and ligated into pGreen-puro vector, while shRNAs targeting HuR and XIRP1 were cloned into pLKO.1-neo vector. Empty vector and scrambled sequence were used as negative controls for overexpression and shRNAs respectively..

搜索）。将靶向circPLOD2a/b和HIF1α的相应寡核苷酸退火并连接到pGreen-puro载体中，同时将靶向HuR和XIRP1的shRNA克隆到pLKO.1-neo载体中。空载体和乱序序列分别用作过表达和shRNA的阴性对照。。

To establish luciferase-labeled cells, a plasmid expressing luciferase and the BSD gene was transfected into U87 cells, followed by selection with Blasticidin S HCl.

为了建立荧光素酶标记的细胞，将表达荧光素酶和BSD基因的质粒转染到U87细胞中，然后用杀稻瘟素S HCl进行选择。

All the primers were purchased from GeneCreate (China), and the sequences are detailed in Supplementary Table

所有引物均购自GeneCreate（中国），序列详见补充表

and Supplementary Table

和补充表

. The expression plasmids, packaging plasmid containing gag-pol and envelope plasmids containing VSVG were co-transfected into HEK293T cells by using Lipofectamine 3000 (Invitrogen, USA) following the manufacturer’s instructions. Lentivirus were collected after 72 h and used for transfection in U87 or LN229 cells.

按照制造商的说明，使用Lipofectamine 3000（Invitrogen，USA）将表达质粒，含有gag-pol的包装质粒和含有VSVG的包膜质粒共转染到HEK293T细胞中。72小时后收集慢病毒并用于U87或LN229细胞中的转染。

Puromycin or neomycin (G418) was used to select the stable cell lines according to the antibiotic resistance genes carried by the plasmids..

嘌呤霉素或新霉素（G418）用于根据质粒携带的抗生素抗性基因选择稳定的细胞系。。

Western blotting

西方墨点法

GBM cells were collected and lysed in RIPA lysis buffer (Beyotime, China). The protein concentrations were determined using a BCA Protein Assay Kit (Beyotime, China). Proteins were separated by 8–12% SDS-PAGE and then transferred to PVDF membranes (Millipore, Schwalbach, Germany). The membrane was blocked with PBST buffer containing 5% skim milk and incubated with the corresponding primary antibodies at 4 °C overnight.

收集GBM细胞并在RIPA裂解缓冲液（Beyotime，China）中裂解。使用BCA蛋白质测定试剂盒（Beyotime，China）测定蛋白质浓度。通过8-12%SDS-PAGE分离蛋白质，然后转移到PVDF膜（Millipore，Schwalbach，德国）上。用含有5%脱脂奶的PBST缓冲液封闭膜，并在4℃下与相应的一抗孵育过夜。

Then, the membrane was washed and hybridized with an HRP-conjugated secondary antibody at room temperature for 2 h. The signals were detected using ChemiDoc XRS+ (Bio-Rad, USA)..

然后，洗涤膜并在室温下与HRP缀合的二抗杂交2小时。使用ChemiDoc XRS+（Bio-Rad，USA）检测信号。。

β-actin was used as a protein internal control due to its independence from hypoxia and HuR regulation. The primary antibodies used for western blotting were anti-β-actin antibody (66009-1-Ig, Proteintech, 1:2000), anti-HIF1α antibody (ab179483, Abcam, 1:1000), anti-PLOD2 antibody (66342-1-Ig, Proteintech, 1:2000), anti-ALDA antibody (11217-1-AP, Proteintech, 1:2000), anti-BIP antibody (11587-1-AP, Proteintech, 1:2000), anti-DDX5 antibody(67025-1-Ig, Proteintech, 1:2000), anti-HuR antibody (11910-1-AP, Proteintech, 1:2000), anti-IGF2BP3 antibody (14642-1-AP, Proteintech, 1:2000), anti-E-cadherin antibody (20874-1-AP, Proteintech, 1:1000), anti-N-cadherin antibody (22018-1-AP, Proteintech, 1:1000), anti-Vimentin antibody (10366-1-AP, Proteintech, 1:1000), anti-Flag antibody (20543-1-AP, Proteintech, 1:2000), anti-XIRP1 antibody(PA5-48605, Invitrogen, 1:1000), HRP Goat anti-Rabbit antibody (AS014, Abclonal, 1:5000), and HRP Goat anti-Mouse antibody (SA00001-1, Proteintech, 1:5000)..

。用于蛋白质印迹的一抗是抗β-肌动蛋白抗体（66009-1-Ig，Proteintech，1:2000），抗HIF1α抗体（ab179483，Abcam，1:1000），抗PLOD2抗体（66342-1-Ig，Proteintech，1:2000），抗ALDA抗体（11217-1-AP，Proteintech，1:2000），抗BIP抗体（11587-1-AP，Proteintech，1:2000），抗DDX5抗体（67025-1-Ig，Proteintech，1:2000），抗HuR抗体（11910-1-AP，Proteintech Intech，1:2000），抗IGF2BP3抗体（14642-1-AP，Proteintech，1:2000），抗E-钙粘蛋白抗体（20874-1-AP，Proteintech，1:1000），抗N-钙粘蛋白抗体（22018-1-AP，Proteintech，1:1000），抗波形蛋白抗体（10366-1-AP，Proteintech，1:1000），抗Flag抗体（20543-1-AP，Proteintech，1:2000），抗XIRP1抗体（PA5-48605，Invitrogen，1:1000），HRP山羊抗兔抗体（PA5-48605，Invitrogen，1:1000）AS014，Abclonal，1:5000）和HRP山羊抗小鼠抗体（SA00001-1，Proteintech，1:5000）。。

Cell viability analysis

细胞活力分析

Cell viability was detected by Cell Counting Kit 8 assays (GLPBIO, USA). 4 * 10

。4*10

cells per well were plated into 96-well plate, and incubated in normoxic condition for 8 h. Then the plates were transferred into hypoxic chamber and treated for 24 h or 48 h. Finally, CCK8 reagent was added to cell medium for absorbance detection according to the manufacturer’s guidelines.

将每孔细胞接种到96孔板中，并在常氧条件下孵育8小时。然后将板转移到低氧室中并处理24小时或48小时。最后，将CCK8试剂加入细胞培养基中根据制造商的指导进行吸光度检测。

Transwell assay

Transwell分析

Transwell chambers were used in migration assays and chambers precoated with 10% Matrigel (Corning, USA) were used in invasion assays. For each replicate, 1 * 10

Transwell小室用于迁移测定，预涂有10%Matrigel（美国康宁）的小室用于侵袭测定。对于每个复制，1*10

cells were plated in 200 μL of serum-free medium on upper chambers which were inserted into a 24-well plate, and 500 μL of medium containing 10% FBS was added to the bottom chamber. After incubation for 24 h under hypoxia, upper chamber cells were gently removed, and the invaded cells in the lower filters were fixed with 4% polymethanol for 10 min, followed by staining with crystal violet for 10 min.

将细胞接种在200μL无血清培养基中的上室中，将其插入24孔板中，并将500μL含有10%FBS的培养基加入底室中。。

The experiments were performed in triplicate and more than five photos of each replicate were taken by Leica DMi8 microscope (Leica, Germany). Cell counting was conducted using ImageJ (version 1.53q, USA)..

实验一式三份进行，每个重复的五张以上照片由Leica DMi8显微镜（Leica，Germany）拍摄。使用ImageJ（版本1.53q，USA）进行细胞计数。。

Wound-healing assay

伤口愈合测定

For wound-healing assay, cells were seeded in 6-well plate at a concentration of 4 * 10

对于伤口愈合测定，将细胞以4×10的浓度接种在6孔板中

/well. After 24 h, 10 μL pipet tip was used to create a wound scratch. Cells were cultured in 1% O

/嗯。24小时后，使用10μL移液管尖端产生伤口划痕。细胞在1%O培养

hypoxic condition for 24 h with serum-free medium. The migration of cells at the wound site was measure by taking images of the wound site at 0 h and 24 h post the wound scratch. ImageJ (version 1.53q, USA) was used to analyze the percentage of wound closure. The experiments were performed in triplicate and >5 photos of each replicate were taken by Leica DMi8 microscope (Leica, Germany)..

用无血清培养基缺氧24小时。通过在伤口划伤后0小时和24小时拍摄伤口部位的图像来测量伤口部位细胞的迁移。ImageJ（版本1.53q，USA）用于分析伤口闭合的百分比。实验一式三份进行，每个重复的>5张照片由Leica DMi8显微镜（Leica，Germany）拍摄。。

Fluorescence in situ hybridization (FISH) and Immunofluorescence staining (IF)

荧光原位杂交（FISH）和免疫荧光染色（IF）

Fluorescence in situ hybridization (FISH) was performed as the manufacturer’s guidelines of Fluorescent in Situ Hybridization Kit (RiboBio, China). Briefly, 2 * 10

。简而言之，2*10

of U87 cells were seeded on a cover clip with a diameter of 14 mm in 24-well plate and treated in normoxia or hypoxia condition. 48 h after treatment, cells were fixed by 4% polymethanol and then incubated with biotin-labeled circPLOD2b probe, which was designed and synthesized by RiboBio (Guangzhou, China).

。处理48小时后，将细胞用4%聚甲醇固定，然后与生物素标记的circPLOD2b探针孵育，该探针由RiboBio（中国广州）设计和合成。

Product number for the specific FISH probe is lnc1100936 Ribo.

特定FISH探针的产品编号为lnc1100936 Ribo。

TM公司

h-hsa_circ_0067682_FISH Probe Mix (Red, 20 T), and the ordering URL is:

h-hsa\u circ\u 0067682\u FISH探针混合物（红色，20T），订购URL为：

https://www.ribobio.com/product-and-service/gene-ncrna-mechanism/fish-probes/

. Nuclei were stained by DAPI. For dual RNA-FISH and immunofluorescent staining, immunofluorescence staining (IF) was conducted after FISH. Briefly, after performing RNA-FISH, the slides were blocked with 2% BSA and incubated with HuR antibody at a dilution of 1:200 (11910-1-AP, Proteintech), followed by incubation with 488 goat anti-rabbit IgG (H + L) (A-11008, Invitrogen, 1:1000).

细胞核被DAPI染色。对于双重RNA-FISH和免疫荧光染色，在FISH后进行免疫荧光染色（IF）。简而言之，在进行RNA-FISH后，将载玻片用2%BSA封闭，并与HuR抗体以1:200的稀释度孵育（11910-1-AP，Proteintech），然后与488山羊抗兔IgG孵育（H + L）（a-11008，Invitrogen，1:1000）。

Images were acquired using Andor Revolution WD spinning disc confocal microscope (Oxford Instruments, England)..

使用Andor Revolution WD旋转圆盘共聚焦显微镜（英国牛津仪器公司）获取图像。。

RNA pull-down and mass spectrometry

RNA下拉和质谱

RNA pull-down assay was carried out with Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, USA) following the manufacturer’s instructions. The biotin-labeled probes targeting the junction site of circPLOD2a and circPLOD2b were synthesized by Genecreate (China) and the sequences are detailed in Supplementary Table .

按照制造商的说明，使用Pierce™磁性RNA蛋白下拉试剂盒（Thermo Fisher Scientific，USA）进行RNA下拉测定。靶向circPLOD2a和circPLOD2b连接位点的生物素标记探针由Genecreate（China）合成，序列详见补充表。

. Briefly, streptavidin magnetic beads were incubated with circPLOD2a-specific or circPLOD2b-specific probes, and then incubated with U87 cell lysate. The product was separated by SDS PAGE. Silver staining was performed to observe the differences between the proteins pulled down by scrambled control and circPLOD2a/b probes.

简而言之，将链霉亲和素磁珠与circPLOD2a特异性或circPLOD2b特异性探针孵育，然后与U87细胞裂解物孵育。通过SDS PAGE分离产物。进行银染以观察通过乱序对照和circPLOD2a/b探针下拉的蛋白质之间的差异。

Each lane of gel was cut and analyzed by mass spectrometry at Genecreate (China). The proteins pulled down were further verified by western blot with anti-ALDA antibody (11217-1-AP, Proteintech, 1:2000), anti-BIP antibody (11587-1-AP, Proteintech, 1:2000), anti-DDX5 antibody (67025-1-Ig, Proteintech, 1:2000), anti-HuR antibody (11910-1-AP, Proteintech, 1:2000) and anti-IGF2BP3 antibody (14642-1-AP, Proteintech, 1:2000)..

在Genecreate（中国）切割凝胶的每个泳道并通过质谱分析。用抗ALDA抗体（11217-1-AP，Proteintech，1:2000），抗BIP抗体（11587-1-AP，Proteintech，1:2000），抗DDX5抗体（67025-1-Ig，Proteintech，1:2000），抗HuR抗体（11910-1-AP，Proteintech，1:2000）和抗IGF2BP3抗体（14642-1-AP，Proteintech，1:2000）通过蛋白质印迹进一步验证下拉的蛋白质。。

RNA immunoprecipitation (RIP)

RNA免疫沉淀（RIP）

RNA immunoprecipitation assay was performed as previously described

如前所述进行RNA免疫沉淀测定

. Briefly, U87 cells were crosslinked with 1% formaldehyde. Cell lysates were collected in RIPA buffer and sonicated using the Bioruptor Plus (Diagenode, Belgium) at 4 °C in high-power mode for 8 cycles (30 s ON, 30 s OFF). The lysates were then centrifuged at 12,000 rpm at 4 °C for 10 min, and the supernatant was diluted with lysis buffer and incubated with antibodies specific for HuR (11910-1-AP, Proteintech, 1:100) or IgG control (30000-0-AP, Proteintech, 1:500) with 100 unites of RNase inhibitor, followed by rotating for 10-14 h at 4 °C.

。将细胞裂解物收集在RIPA缓冲液中，并使用Bioruptor Plus（Diagenode，Belgium）在4°C的高功率模式下超声处理8个循环（30 s，30秒关闭）。然后将裂解物在4℃以12000 rpm离心10分钟，并用裂解缓冲液稀释上清液，并与对HuR（11910-1-AP，Proteintech，1:100）或IgG特异性抗体孵育。对照（30000-0-AP，Proteintech，1:500）和100单位的RNase抑制剂，然后在4℃旋转10-14小时。

Mix of Dynabeads protein A (10001D, Invitrogen, USA) and Dynabeads protein G (10003D, Invitrogen, USA) were used to immunoprecipitated protein and associate RNA. The RNA-protein-beads complex and input sample were digested by 500 μg/mL protein kinase K at 55 °C for 1 h, followed by retro-crosslinking with 100 mM Tris-HCl (pH = 7.4), 1% SDS and 200 mM NaCl at 70 °C for 10-12 h.

将Dynabeads蛋白A（10001D，Invitrogen，USA）和Dynabeads蛋白G（10003D，Invitrogen，USA）的混合物用于免疫沉淀蛋白和缔合RNA。将RNA-蛋白珠复合物和输入样品在55°C下用500μG/mL蛋白激酶K消化1小时，然后用100 mM Tris-HCl（pH = 7.4), 。

The co-precipitated RNAs were extracted by TRIZOL and detected by qRT-PCR. Immunoprecipitated proteins were boiled at 99 °C for 10 min and separated by western blot. For circPLOD2a/b and HuR mapping assays, plasmids carrying Flag-tagged HuR (wt/.

通过TRIZOL提取共沉淀的RNA，并通过qRT-PCR检测。。对于circPLOD2a/b和HuR作图测定，携带Flag标记的HuR（wt/）的质粒。

△

RRM1/2/3) were transiently transfected into U87 using Lipofectamine 3000 (Invitrogen, USA). Anti-Flag Magnetic Beads (Beyotime, China) was used for RNA immunoprecipitation. Anti-Flag antibody (20543-1-AP, Proteintech, 1:200) was used to detect the immunoprecipitated protein.

使用Lipofectamine 3000（Invitrogen，USA）将RRM1/2/3）瞬时转染到U87中。抗Flag磁珠（Beyotime，China）用于RNA免疫沉淀。使用抗Flag抗体（20543-1-AP，Proteintech，1:200）检测免疫沉淀的蛋白质。

GBM xenograft mouse model

GBM异种移植小鼠模型

The animal study was performed after receiving approval from the Institutional Animal Care and Use Committee of Hubei University of Medicine (approval number: 2023008).

动物研究是在获得湖北医科大学机构动物护理和使用委员会（批准号：2023008）批准后进行的。

Four-week-old male BALB/c nude mice were purchased from Beijing Vitalstar Biotechnology Co. Ltd. (China) and housed under the standard conditions at the Center of Experimental Animals of Hubei University of Medicine. Mice were randomly allocated into different groups for luciferase-labeled (-luc) U87 cell injection (control-luc, KD-circPLOD2a-luc, KD-circPLOD2a/KD-.

四周大的雄性BALB/c裸鼠购自北京维塔尔生物技术有限公司（中国），并在标准条件下饲养在湖北医科大学实验动物中心。将小鼠随机分为不同组进行荧光素酶标记的（-luc）U87细胞注射（对照luc，KD-circPLOD2a-luc，KD-circPLOD2a/KD-）。

XIRP1

-luc, KD-circPLOD2b-luc, KD-circPLOD2b/KD-

-luc，KD-circPLOD2b-luc，KD-circPLOD2 b/KD-

XIRP1

-luc). For the intracranial xenografts, mice were anesthetized and placed in a stereotact, and 5 * 10

-卢克）。

U87 cells labeled with luciferase were intracranially injected at 2 mm lateral and 1 mm anterior to bregma, 2 mm below the skull into the brain of randomly grouped nude mice. To image the tumor xenografts, the mice were anesthetized using isoflurane and intraperitoneally injected with 150 mg/kg D-luciferin (Beyotime, China) once a week starting at 10 days after the cell injection.

用荧光素酶标记的U87细胞在前囟外侧2毫米和前方1毫米处颅内注射，在头骨下方2毫米处进入随机分组的裸鼠的大脑。为了对肿瘤异种移植物成像，使用异氟烷麻醉小鼠，并从细胞注射后10天开始每周一次腹膜内注射150 mg/kg D-荧光素（Beyotime，China）。

Tumor cells labeled by luminescence were imaged using a spectrum in vivo imaging system (IVIS) (PerkinElmer, USA). Fifty-two days post injection, tumor-bearing mice were humanely sacrificed and brains were harvested and fixed with phosphate-buffered formalin for further immunohistochemistry (IHC) staining..

使用光谱体内成像系统（IVIS）（PerkinElmer，USA）对发光标记的肿瘤细胞进行成像。注射后52天，将荷瘤小鼠人道处死，收获大脑并用磷酸盐缓冲的福尔马林固定以进行进一步的免疫组织化学（IHC）染色。。

We have complied with all relevant ethical regulations for animal use.

我们遵守了动物使用的所有相关道德法规。

Immunohistochemistry staining

免疫组织化学染色

Mice were sacrificed at 52 days post injection and immediately perfused with normal saline and 4% paraformaldehyde solution continuously. The brains were dissected, postfixed for 24 h at 4 °C, and sectioned with a thickness of 10 μm using Leica cryostat microtome (Leica CM1860, Germany). For immunohistochemistry, the slices were blocked in 2% BSA and then incubated with anti-E-cadherin antibody (20874-1-AP, Proteintech, 1:100), anti-N-cadherin antibody (22018-1-AP, Proteintech, 1:100) and anti-Vimentin antibody (10366-1-AP, Proteintech, 1:100).

注射后52天处死小鼠，立即连续灌注生理盐水和4%多聚甲醛溶液。解剖大脑，在4℃下后固定24小时，并使用Leica低温恒温器切片机（Leica CM1860，Germany）切成10μm的厚度。对于免疫组织化学，将切片用2%BSA封闭，然后与抗E-钙粘蛋白抗体（20874-1-AP，Proteintech，1:100），抗N-钙粘蛋白抗体（22018-1-AP，Proteintech，1:100）和抗波形蛋白抗体（10366-1-AP，Proteintech，1:100）孵育。

HRP Goat anti-Rabbit antibody (AS014, Abclonal, 1:100) was used as the secondary antibody. DAB Horseradish Peroxidase Color Development Kit (Beyotime, China) was used as chromogenic reagent. Nuclei were stained by hematoxylin. More than five photos of each group were taken by Leica DMi8 microscope (Leica, Germany)..

HRP山羊抗兔抗体（AS014，Abclonal，1:100）用作二抗。DAB辣根过氧化物酶显色试剂盒（Beyotime，China）用作显色试剂。细胞核被苏木精染色。徕卡DMi8显微镜（德国徕卡）拍摄了每组五张以上的照片。。

Human tissue samples

人体组织样本

Tumor tissues from patients with grade II-III (

II-III级患者的肿瘤组织(

= 10) and IV (

(

= 9) glioma used for qRT-PCR were collected from Taihe Hospital, Hubei, China. Informed consents were obtained from glioma patients for sample collection, and the study was approved by the Ethics Committee of Shiyan Taihe Hospital (approval number: 2023KS11). All tissues were preserved in liquid nitrogen immediately after the surgery at Taihe Hospital, Hubei, China.

9）用于qRT-PCR的神经胶质瘤来自中国湖北省太和医院。。手术后立即在中国湖北省太和医院将所有组织保存在液氮中。

Patient information is shown in Supplementary Table .

患者信息显示在补充表中。

A tissue microarray (TMA) containing grade I (

含有I级的组织微阵列（TMA）(

= 3), II (

= 3），ii（

= 50), III (

= 50），三（

= 24) and IV (

24）和IV(

= 94) glioma tissues was purchased from Zhuoli Biotechnology (ZL-BraGsur1801, Zhuoli Biotechnology Co., Shanghai, China). The patient information with tumor grade and survival time is shown in Supplementary Tables

94）神经胶质瘤组织购自Zhuoli Biotechnology（ZL-BraGsur1801，Zhuoli Biotechnology Co.，Shanghai，China）。肿瘤分级和生存时间的患者信息见补充表

respectively.

分别。

IHC staining of tissue microarray was performed to evaluate the expression level of XIRP1 in these patients. Generally, TMA was incubated with anti-XIRP1 antibody (PA5-48605, Invitrogen, 1:10) for immunohistochemistry staining. By using the Arrayimager software module from Visiopharm (Hoersholm, Denmark), individual digital images of each core was automatically extracted from the full slide images of the TMA.

。通常，将TMA与抗XIRP1抗体（PA5-48605，Invitrogen，1:10）孵育以进行免疫组织化学染色。通过使用Visiopharm（丹麦Hoersholm）的Arrayimager软件模块，从TMA的完整幻灯片图像中自动提取每个核心的单个数字图像。

Intensity of staining (weak/ moderate/ strong) was measured to evaluated the expression of XIRP1 in each cell of these tissues. Histochemistry score (H-score) was calculated to represent the expression level of XIRP1 for each tissue in microarray. [H-Score = ∑ (pi×i) = (percentage of weak intensity×1) + (percentage of moderate intensity×2) + (percentage of strong intensity × 3)]..

测量染色强度（弱/中/强）以评估XIRP1在这些组织的每个细胞中的表达。计算组织化学评分（H评分）以代表微阵列中每个组织的XIRP1表达水平。[H分数=∑（pi×i）=（弱强度百分比×1）+（中等强度百分比×2）+（强强度百分比×3）]。。

All ethical regulations relevant to human research participants were followed.

遵循与人类研究参与者相关的所有道德法规。

Dual-luciferase reporter assay

双荧光素酶报告基因测定

Human

人类

PLOD2

promoter from NC_000003.12:c146163185-146161185 were amplified from 293 T gDNA and subcloned into pGL3-basic to construct luciferase reporter vectors. Cells expressing HIF1α were co-transfected with pGL3-basic empty vector or pGL3-basic-PLOD2 reporter constructs and renilla luciferase plasmids. The luciferase activity of the reporters was detected after 48 h post transfection with Dual-Lumi II Luciferase Assay Kit (Beyotime, China) and normalized to renilla luciferase activity..

来自NC 00000 3.12的启动子：从293T gDNA扩增c146163185-146161185，并亚克隆到pGL3-basic中以构建荧光素酶报告载体。将表达HIF1α的细胞与pGL3-basic空载体或pGL3-basic-PLOD2报告基因构建体和海肾荧光素酶质粒共转染。用Dual Lumi II荧光素酶测定试剂盒（Beyotime，China）转染后48小时检测报告基因的荧光素酶活性，并将其标准化为海肾荧光素酶活性。。

Sucrose gradient ribosome separation assay

蔗糖梯度核糖体分离测定

Ribosomes from U87 KD-HuR and control cells were separated and analyzed following previously established protocols

按照先前建立的方案分离并分析了U87 KD HuR和对照细胞的核糖体

. Briefly, a sucrose gradient solution was prepared in ultracentrifuge tubes with layers at concentrations of 10%, 20%, 30%, 40%, and 50%, containing 100 mM NaCl, 20 mM Tris-HCl (pH 7.5), and 5 mM MgCl

简而言之，在超速离心管中制备蔗糖梯度溶液，其浓度为10%，20%，30%，40%和50%，含有100mM NaCl，20mM Tris-HCl（pH 7.5）和5mM MgCl

. Cells were incubated with 100 μg/mL cycloheximide (GC17198, GLPBIO, USA) for 10 min and washed by pre-cold PBS also contain 100 μg/mL cycloheximide. Cell lysates were prepared in 450 μL of PEB buffer (20 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl

将细胞与100μg/mL环己酰亚胺（GC17198，GLPBIO，USA）孵育10分钟，并用也含有100μg/mL环己酰亚胺的预冷PBS洗涤。在450μlPEB缓冲液（20mM Tris-HCl（pH 7.5））中制备细胞裂解物，100毫米KCl，5毫米MgCl

, 0.5% Nonidet P-40, 100 μg/mL cycloheximide, protease inhibitors, and 100 units of RNase inhibitor). 120 μg of RNA was determined by NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) and layered on top of the sucrose gradient solution. The mixture was centrifuged for 120 min at 190,000 g and 4 °C in a SW41Ti swinging-bucket rotor using a Beckman ultracentrifuge, with maximum acceleration and brake applied.

，0.5%Nonidet P-40，100微克/毫升环己酰亚胺，蛋白酶抑制剂和100单位的RNase抑制剂）。通过NanoDrop 2000分光光度计（Thermo Fisher Scientific，USA）测定120μgRNA，并将其铺在蔗糖梯度溶液的顶部。使用Beckman超速离心机将混合物在SW41Ti摆动桶转子中以190000 g和4℃离心120分钟，施加最大加速度和制动器。

Polysome profiling was analyzed using a piston gradient fractionator (153-002, BioComp, Canda). Ten polysome fractions were sequentially collected from the top to the bottom of the gradient. Total RNA was extracted using TRIzol, and qRT-PCR was performed to measure the expression of HuR and XIRP1 in each fraction.

使用活塞梯度分馏器（153-002，BioComp，Canda）分析多核糖体分析。。使用TRIzol提取总RNA，并进行qRT-PCR以测量每个级分中HuR和XIRP1的表达。

β-actin served as the internal control..

β-肌动蛋白作为内部对照。。

Statistics and reproducibility

统计和再现性

Data are shown as the mean ± SEM (error bars) of at least three independent experiments. Statistical analyses were performed with GraphPad Prism 9.0 (GraphPad Prism, USA). Student’s

数据显示为至少三次独立实验的平均SEM（误差棒）。使用GraphPad Prism 9.0（GraphPad Prism，USA）进行统计分析。学生的

-test or analysis of variance (ANOVA) were applied in the comparison of differences between groups. Survival curves were plotted by Kaplan–Meier method and assessed by log-rank test.

-检验或方差分析（ANOVA）用于比较组间差异。通过Kaplan–Meier方法绘制生存曲线，并通过对数秩检验进行评估。

Reporting summary

报告摘要

Further information on research design is available in the

Nature Portfolio Reporting Summary

自然投资组合报告摘要

linked to this article.

链接到本文。

Data availability

数据可用性

RNA-seq data were deposited in GEO accession GSE245635, GSE245636 and GSE226538. Other data that support the findings of this study are available from the corresponding author upon reasonable request. The source data behind the graphs in the paper can be found in Supplementary Data

RNA-seq数据保存在GEO登录号GSE245635，GSE245636和GSE226538中。根据合理的要求，通讯作者可以提供支持本研究结果的其他数据。本文图表背后的源数据可以在补充数据中找到

. The uncropped images of western blots can be found in

。未剪切的蛋白质印迹图像可以在

Supplementary Information

补充信息

. Newly generated plasmids production and their reference sequences have been deposited in Addgene (pLKO.1-neo-shHuR, ID: 232044; pLCDH-circPLOD2a, ID: 232045; pLCDH-circPLOD2b, ID: 232046; pGreen-puro-circPLOD2a-sh1#, ID: 232047; pGreen-puro-circPLOD2a-sh2#, ID: 232048; pGreen-puro-circPLOD2b-sh1#, ID: 232049; pGreen-puro-circPLOD2b-sh2#, ID: 232050; pLKO.1-neo-shXIRP1, ID: 232051; pGreen-puro-HIF1α-sh1#, ID: 232052; pGL3-Basic-PLOD2, ID: 232053; pGreen-puro-HIF1α-sh2#, ID: 232054)..

。新产生的质粒生产及其参考序列已保存在Addgene中（pLKO.1-neo-shHuR，ID:232044；pLCDH-circPLOD2a，ID:232045；pLCDH-circPLOD2b，ID:232046；pGreen-puro-circPLOD2a-sh1#，ID:232047；pGreen-puro-circPLOD2a-sh2#，ID:232048；pGreen-puro-circPLOD2b-sh1#，ID:232049；pGreen-puro-circPLOD2b-sh2#，ID:232050；pLKO.1-neo-shXIRP1，ID:232051；pGreen-puro-HIF1α-sh1#，ID:232052；pGL3-Basic-PLOD2，ID:232053；pGreen-puro-HIF1α-sh2#，ID:232054）。。

References

参考文献

Lapointe, S., Perry, A. & Butowski, N. A. Primary brain tumours in adults.

Lapointe，S.，Perry，A。＆Butowski，N.A。成人原发性脑肿瘤。

Lancet

柳叶刀

392

, 432–446 (2018).

Article

文章

PubMed

Google Scholar

谷歌学者

Ortiz-Prado, E., Dunn, J. F., Vasconez, J., Castillo, D. & Viscor, G. Partial pressure of oxygen in the human body: a general review.

。

Am. J. Blood Res.

Am.J.Blood Res。

, 1–14 (2019).

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Muz, B., de la Puente, P., Azab, F. & Azab, A. K. The role of hypoxia in cancer progression, angiogenesis, metastasis, and resistance to therapy.

。

Hypoxia (Auckl)

缺氧（Auckl）

, 83–92 (2015).

Article

文章

PubMed

Google Scholar

谷歌学者

Noman, M. Z. et al. PD-L1 is a novel direct target of HIF-1 alpha., and its blockade under hypoxia enhanced MDSC-mediated T cell activation.

Noman，M.Z.等人PD-L1是HIF-1α的新型直接靶标。，缺氧条件下的阻断增强了MDSC介导的T细胞活化。

J. Exp. Med.

实验医学学报

211

, 781–790 (2014).

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Peng, G. et al. The HIF1 alpha-PDGFD-PDGFR alpha axis controls glioblastoma growth at normoxia/mild-hypoxia and confers sensitivity to targeted therapy by echinomycin.

Peng，G。等人。HIF1α-PDGFD-PDGFRα轴在常氧/轻度缺氧条件下控制胶质母细胞瘤的生长，并赋予棘霉素靶向治疗的敏感性。

J. Exp. Clin. Cancer Res.

J、实验临床。癌症研究。

, 16 (2021).

Article

文章

Google Scholar

谷歌学者

Joseph, J. V. et al. Hypoxia enhances migration and invasion in glioblastoma by promoting a mesenchymal shift mediated by the HIF1 alpha-ZEB1 axis.

Joseph，J.V.等人。缺氧通过促进由HIF1α-ZEB1轴介导的间充质转变来增强胶质母细胞瘤的迁移和侵袭。

Cancer Lett.

癌症床。

359

, 107–116 (2015).

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Goldberg, M. A., Dunning, S. P. & Bunn, H. F. Regulation of the erythropoietin gene: evidence that the oxygen sensor is a heme protein.

Goldberg，M.A.，Dunning，S.P。＆Bunn，H.F。促红细胞生成素基因的调节：氧传感器是血红素蛋白的证据。

Science

科学

242

, 1412–1415 (1988).

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Ma, Z. W. et al. Targeting hypoxia-inducible factor-1-mediated metastasis for cancer therapy.

Ma，Z.W.等。靶向缺氧诱导因子-1介导的转移用于癌症治疗。

Antioxid Redox Signal

抗氧化氧化还原信号

, 1484–1497 (2021).

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Mendez, O. et al. Knock down of HIF-1 alpha in glioma cells reduces migration in vitro and invasion in vivo and impairs their ability to form tumor spheres.

敲除胶质瘤细胞中的HIF-1α可减少体外迁移和体内侵袭，并削弱其形成肿瘤球的能力。

Mol. Cancer

分子癌症

, 10 (2010).

Article

文章

Google Scholar

谷歌学者

Liu, C. X. & Chen, L. L. Circular RNAs: characterization, cellular roles, and applications.

Liu，C.X。＆Chen，L.L。环状RNA：表征，细胞作用和应用。

Cell

细胞

185

, 2016–2034 (2022).

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Kristensen, L. S. et al. The biogenesis, biology and characterization of circular RNAs.

Kristensen，L.S.等人。环状RNA的生物发生，生物学和表征。

Nat. Rev. Genet.

《遗传学》杂志。

, 675–691 (2019).

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Meng, S. J. et al. CircRNA: functions and properties of a novel potential biomarker for cancer.

Meng，S.J.等人。CircRNA：一种新型潜在癌症生物标志物的功能和特性。

Mol. Cancer

分子癌症

, 8 (2017).

Article

文章

Google Scholar

谷歌学者

Yang, R. et al. Hypoxia-induced circWSB1 promotes breast cancer progression through destabilizing p53 by interacting with USP10.

Yang，R。等人。缺氧诱导的circWSB1通过与USP10相互作用破坏p53的稳定性来促进乳腺癌的进展。

Mol. Cancer

分子癌症

, 88 (2022).

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Yang, H. O. et al. Hypoxia induced exosomal circRNA promotes metastasis of colorectal cancer via targeting GEF-H1/RhoA axis.

Yang，H.O.等人。缺氧诱导的外泌体circRNA通过靶向GEF-H1/RhoA轴促进结直肠癌的转移。

Theranostics

治疗诊断科技

, 8211–8226 (2020).

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Wang, R. et al. CircNT5E acts as a sponge of miR-422a to promote glioblastoma tumorigenesis.

Wang，R。等人。CircNT5E充当miR-422a的海绵以促进胶质母细胞瘤肿瘤发生。

Cancer Res.

癌症研究。

, 4812–4825 (2018).

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Du, W. W. et al. Identifying and characterizing circRNA-protein interaction.

Du，W。W。等人。鉴定和表征circRNA-蛋白质相互作用。

Theranostics

治疗诊断科技

, 4183–4191 (2017).

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Xia, X. et al. A novel tumor suppressor protein encoded by circular AKT3 RNA inhibits glioblastoma tumorigenicity by competing with active phosphoinositide-dependent Kinase-1.

Xia，X。等人。一种由环状AKT3 RNA编码的新型肿瘤抑制蛋白通过与活性磷酸肌醇依赖性激酶-1竞争来抑制胶质母细胞瘤的致瘤性。

Mol. Cancer

分子癌症

, 16 (2019).

Google Scholar

谷歌学者

Srikantan, S. Gorospe M. HuR function in disease.

Srikantan，S.Gorospe M.HuR在疾病中的功能。

Front. Biosci.

前面生物。

, 189–205 (2012).

Article

文章

CAS

中科院

Google Scholar

谷歌学者

Doller, A. et al. Posttranslational modification of the AU-rich element binding protein HuR by protein kinase C delta elicits angiotensin II-induced stabilization and nuclear export of clooxygenase 2 mRNA.

Doller，A。等人。蛋白激酶Cδ对富含AU的元件结合蛋白HuR的翻译后修饰引起血管紧张素II诱导的环氧合酶2 mRNA的稳定和核输出。

Mol. Cell Biol.

分子细胞生物学。

, 2608–2625 (2008).

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Brennan, C. M. Steitz JA. HuR and mRNA stability.

。HuR和mRNA稳定性。

Cell. Mol. Life Sci.

细胞。分子生命科学。

, 266–277 (2001).

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Chen, J. et al. Circular RNA circRHOBTB3 represses metastasis by regulating the HuR-mediated mRNA stability of PTBP1 in colorectal cancer.

Chen，J。等人。环状RNA circRHOBTB3通过调节结直肠癌中HuR介导的PTBP1 mRNA稳定性来抑制转移。

Theranostics

治疗诊断科技

, 7507–7526 (2021).

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Zhu, T. et al. Oncogenic circTICRR suppresses autophagy via binding to HuR protein and stabilizing GLUD1 mRNA in cervical cancer.

Zhu，T。等人。致癌circTICRR通过与HuR蛋白结合并稳定宫颈癌中的GLUD1 mRNA来抑制自噬。

Cell Death Dis.

细胞死亡Dis。

, 479 (2022).

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Song, X. et al. CircEIF3H-IGF2BP2-HuR scaffold complex promotes TNBC progression via stabilizing HSPD1/RBM8A/G3BP1 mRNA.

Song，X。等人。CircEIF3H-IGF2BP2-HuR支架复合物通过稳定HSPD1/RBM8A/G3BP1 mRNA促进TNBC进展。

Cell Death Discov.

细胞死亡发现。

, 261 (2022).

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Abdelmohsen, K. et al. Identification of HuR target circular RNAs uncovers suppression of PABPN1 translation by CircPABPN1.

Abdelmohsen，K。等人。HuR靶环状RNA的鉴定揭示了CircPABPN1对PABPN1翻译的抑制。

RNA Biol.

RNA生物。

, 361–369 (2017).

Article

文章

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Okumura, Y. et al. Hypoxia-induced PLOD2 is a key regulator in epithelial-mesenchymal transition and chemoresistance in Biliary tract cancer.

Okumura，Y.等人。缺氧诱导的PLOD2是胆管癌上皮-间质转化和化疗耐药的关键调节因子。

Ann. Surg. Oncol.

安。外科。Oncol。

, 3728–3737 (2018).

Article

文章

PubMed

Google Scholar

谷歌学者

Kresse, N. et al. PLOD2 is a prognostic marker in glioblastoma that modulates the immune microenvironment and tumor progression.

PLOD2是胶质母细胞瘤的预后标志物，可调节免疫微环境和肿瘤进展。

Int J Mol Sci

国际分子科学杂志

, 6037 (2022).

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Du, H., Pang, M., Hou, X., Yuan, S. & Sun, L. PLOD2 in cancer research.

Du，H.，Pang，M.，Hou，X.，Yuan，S。＆Sun，L。PLOD2在癌症研究中。

Biomed. Pharmacother.

生物医学。药剂师。

, 670–676 (2017).

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Noda, T. et al. PLOD2 induced under hypoxia is a novel prognostic factor for hepatocellular carcinoma after curative resection.

缺氧诱导的PLOD2是肝细胞癌根治性切除术后的一种新的预后因素。

Ann. Surg. Oncol.

安。外科。Oncol。

, S121–S121 (2012).

，S121–S121（2012）。

Google Scholar

谷歌学者

Lal, A. et al. Transcriptional response to hypoxia in human tumors.

Lal，A。等人。人类肿瘤对缺氧的转录反应。

JNCI J. Natl Cancer Institute

JNCI J.Natl癌症研究所

, 1337–1343 (2001).

Article

文章

CAS

中科院

Google Scholar

谷歌学者

Sun, J. et al. A novel lncRNA ARST represses glioma progression by inhibiting ALDOA-mediated actin cytoskeleton integrity.

Sun，J。等人。一种新型lncRNA-ARST通过抑制ALDOA介导的肌动蛋白细胞骨架完整性来抑制神经胶质瘤的进展。

J. Exp. Clin. Cancer Res.

J、实验临床。癌症研究。

, 20 (2021).

Article

文章

Google Scholar

谷歌学者

Li, X. et al. Aldolase A enhances intrahepatic cholangiocarcinoma proliferation and invasion through promoting glycolysis.

Li，X。等。醛缩酶A通过促进糖酵解增强肝内胆管癌的增殖和侵袭。

Int. J. Biol. Sci.

J. Biol。滑雪。

, 1782–1794 (2021).

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Wang, R. et al. P68 RNA helicase promotes invasion of glioma cells through negatively regulating DUSP5.

Wang，R。等人。P68 RNA解旋酶通过负调节DUSP5促进神经胶质瘤细胞的侵袭。

Cancer Sci.

癌症科学。

110

, 107–117 (2019).

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Nabors, L. L. B. et al. Tumor necrosis factor a induces angiogenic factor up-regulation in malignant glioma cells: a role for RNA stabilization and HuR.

。

Cancer Res.

癌症研究。

, 4181–4187 (2003).

CAS

中科院

PubMed

Google Scholar

谷歌学者

Lebedeva, S. et al. Transcriptome-wide analysis of regulatory interactions of the RNA-binding protein HuR.

。

Mol. Cell

摩尔电池

, 340–352 (2011).

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Lee, D. J. et al. Multiple tumor-suppressor genes on chromosome 3p contribute to head and neck squamous cell carcinoma tumorigenesis.

Lee，D.J.等人。染色体3p上的多个肿瘤抑制基因有助于头颈部鳞状细胞癌的发生。

Cancer Biol. Ther.

癌症生物学。。

, 689–693 (2010).

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Madhavan, S. et al. Rembrandt: helping personalized medicine become a reality through integrative translational research.

Madhavan，S.等人。伦勃朗：通过整合转化研究帮助个性化医学成为现实。

Mol Cancer Res.

摩尔癌症研究。

, 157–167 (2009).

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Hansen, T. B. et al. Natural RNA circles function as efficient microRNA sponges.

Hansen，T.B.等人。天然RNA环作为有效的microRNA海绵起作用。

Nature

自然

495

, 384–388 (2013).

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Li, X. et al. Linking circular intronic RNA degradation and function in transcription by RNase H1.

Li，X。等人。通过RNase H1将环状内含子RNA降解与转录功能联系起来。

Sci China Life Sci.

Sci中国生命科学。

, 1795–1809 (2021).

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Legnini, I. et al. Circ-ZNF609 is a circular RNA that can be translated and functions in myogenesis.

Legnini，I。等人Circ-ZNF609是一种环状RNA，可以翻译并在肌生成中起作用。

Mol. Cell

摩尔电池

, 22–37 (2017).

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Chen, X. P. et al. circRNADb: A comprehensive database for human circular RNAs with protein-coding annotations.

Chen，X。P。等人。circRNADb：具有蛋白质编码注释的人类环状RNA的综合数据库。

Sci. Rep.

科学。代表。

, 6 (2016).

Google Scholar

谷歌学者

Arroyo, J. D. et al. Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma.

Arroyo，J.D。等人，Argonaute2复合物携带大量独立于人血浆中囊泡的循环microRNA。

Proc. Natl Acad. Sci. USA

普罗克。阿卡德之夜滑雪。美国

108

, 5003–5008 (2011).

Article

文章

CAS

中科院

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Du, A. S. et al. M6A-mediated upregulation of circMDK promotes tumorigenesis and acts as a nanotherapeutic target in hepatocellular carcinoma.

Du，A.S.等人，M6A介导的circMDK上调促进肿瘤发生，并作为肝细胞癌的纳米治疗靶点。

Mol. Cancer

分子癌症

, 18 (2022).

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Huang, Y. H., Hong, W. Q. & Wei, X. W. The molecular mechanisms and therapeutic strategies of EMT in tumor progression and metastasis.

Huang，Y.H.，Hong，W.Q。＆Wei，X.W。EMT在肿瘤进展和转移中的分子机制和治疗策略。

J. Hematol. Oncol.

J、赫马托尔。肿瘤。

, 27 (2022).

Article

文章

CAS

中科院

Google Scholar

谷歌学者

Zhang, C. et al. The short inverted repeats-induced circEXOC6B inhibits prostate cancer metastasis by enhancing the binding of RBMS1 and HuR.

Zhang，C。等人。短反向重复序列诱导的circEXOC6B通过增强RBMS1和HuR的结合来抑制前列腺癌转移。

Mol. Ther.

摩尔热。

, 1705–1721 (2023).

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Vaupel, P. & Mayer, A. Hypoxia in cancer: significance and impact on clinical outcome.

。

Cancer Metastasis Rev.

癌症转移Rev。

, 225–239 (2007).

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Higgins, D. F. et al. Hypoxia promotes fibrogenesis in vivo via HIF-1 stimulation of epithelial-to-mesenchymal transition.

Higgins，D.F。等人。缺氧通过HIF-1刺激上皮-间质转化促进体内纤维发生。

J. Clin. Invest.

J、临床。投资。

117

, 3810–3820 (2007).

CAS

中科院

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Conn, V. M. et al. A circRNA from SEPALLATA3 regulates splicing of its cognate mRNA through R-loop formation.

Conn，V.M.等人。来自SEPALLATA3的circRNA通过R环形成调节其同源mRNA的剪接。

Nat. Plants

天然植物

, 5 (2017).

Article

文章

Google Scholar

谷歌学者

Dong, S. M. et al. Histology-based expression profiling yields novel prognostic markers in human glioblastoma.

Dong，S.M.等人。基于组织学的表达谱分析在人类胶质母细胞瘤中产生了新的预后标志物。

J. Neuropathol. Exp. Neurol.

J 神经病理学家Exp. 神经元。

, 948–955 (2005).

Article

文章

PubMed

Google Scholar

谷歌学者

Majumder, M., Chakraborty, P., Mohan, S., Mehrotra, S. & Palanisamy, V. HuR as a molecular target for cancer therapeutics and immune-related disorders.

Majumder，M.，Chakraborty，P.，Mohan，S.，Mehrotra，S。＆Palanisamy，V。HuR作为癌症治疗和免疫相关疾病的分子靶标。

Adv. Drug Deliv. Rev.

高级药物输送。版次：。

188

, 114442 (2022).

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Yang, L. Q. et al. Muscone derivative ZM-32 inhibits breast tumor angiogenesis by suppressing HuR-mediated VEGF and MMP9 expression.

。

Biomed. Pharmacother.

生物医学。药剂师。

136

, 11 (2021).

Article

文章

Google Scholar

谷歌学者

Guha, A. et al. The versatile role of HuR in glioblastoma and its potential as a therapeutic target for a multi-pronged attack.

Guha，A。等人。HuR在胶质母细胞瘤中的多功能作用及其作为多管齐下的治疗靶点的潜力。

Adv. Drug Deliv. Rev.

高级药物输送。版次：。

181

, 13 (2022).

Article

文章

Google Scholar

谷歌学者

Akool, E. S. et al. Nitric oxide increases the decay of matrix metalloproteinase 9 mRNA by inhibiting the expression of mRNA-stabilizing factor HuR.

Akool，E.S.等人。一氧化氮通过抑制mRNA稳定因子HuR的表达来增加基质金属蛋白酶9 mRNA的衰变。

Mol. Cell Biol.

分子细胞生物学。

, 4901–4916 (2003).

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Wang, J. P. et al. Deletion of the RNA regulator HuR in tumor-associated microglia and macrophages stimulates anti-tumor immunity and attenuates glioma growth.

Wang，J.P.等人。肿瘤相关小胶质细胞和巨噬细胞中RNA调节因子HuR的缺失刺激抗肿瘤免疫力并减弱神经胶质瘤的生长。

Glia

神经胶质

, 2424–2439 (2019).

Article

文章

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Gaur, A. B., Holbeck, S. L., Colburn, N. H. & Israel, M. A. Downregulation of Pdcd4 by mir-21 facilitates glioblastoma proliferation in vivo.

Gaur，A.B.，Holbeck，S.L.，Colburn，N.H。和Israel，M.A。mir-21下调Pdcd4促进体内胶质母细胞瘤增殖。

Neuro Oncol.

神经肿瘤学。

, 580–590 (2011).

Article

文章

CAS

中科院

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

Wigington, C. P. et al. Post-transcriptional regulation of programmed cell death 4 (PDCD4) mRNA by the RNA-binding proteins human antigen R (HuR) and T-cell intracellular antigen 1 (TIA1).

。

J. Biol. Chem.

J. 比尔化学。

290

, 3468–3487 (2015).

Article

文章

CAS

中科院

PubMed

Google Scholar

谷歌学者

Casolaro, V. et al. Posttranscriptional regulation of IL-13 in T cells: role of the RNA-binding protein HuR.

Casolaro，V。等人。T细胞中IL-13的转录后调控：RNA结合蛋白HuR的作用。

J. Allergy Clin. Immunol.

J、。免疫。

121

, 853–859 (2008).

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Thaci, B. et al. Significance of interleukin-13 receptor alpha 2-targeted glioblastoma therapy.

Thaci，B。等人。白细胞介素-13受体α2靶向胶质母细胞瘤治疗的意义。

Neuro Oncol.

神经肿瘤学。

, 1304–1312 (2014).

Article

文章

CAS

中科院

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Di Ruscio, A. et al. DNMT1-interacting RNAs block gene-specific DNA methylation.

Di Ruscio，A。等人。DNMT1相互作用的RNA阻断基因特异性DNA甲基化。

Nature

自然

503

, 371–376 (2013).

Article

文章

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Panda, A. C., Martindale, J. L. & Gorospe, M. Polysome fractionation to analyze mRNA distribution profiles.

Panda，A.C.，Martindale，J.L。＆Gorospe，M。Polysome分级以分析mRNA分布谱。

Bio. Protoc.

生物，原因

, e2126 (2017).

，e2126（2017）。

Article

文章

PubMed

PubMed Central

PubMed 中央

Google Scholar

谷歌学者

Zeng, X. et al. CircHIPK2 contributes cell growth in intestinal epithelial of colitis and colorectal cancer through promoting TAZ translation.

Zeng，X。等人。CircHIPK2通过促进TAZ翻译促进结肠炎和结直肠癌肠上皮细胞生长。

Adv. Sci. (Weinh)

高级科学。（温）

, 2401588 (2024).

Article

文章

CAS

中科院

PubMed

PubMed Central

公共医学中心

Google Scholar

谷歌学者

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Acknowledgements

This work was supported by Key Project of Research and Development of Hubei Province (2022BCE049), Natural Science Foundation of Hubei Province (2023AFB1037, 2017CFA064), Fundamental Research Funds for the Central Universities (2662021SYQD001), National Natural Science Foundation of China (82103674)..

这项工作得到了湖北省研究与发展重点项目（2022BCE049），湖北省自然科学基金（2023AFB10372017CFA064），中央大学基础研究基金（2662021SYQD001），国家自然科学基金（82103674）的支持。。

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作者笔记

These authors contributed equally: Wendai Bao, Zhiqiang Dong.

这些作者做出了同样的贡献：鲍文岱，董志强。

Authors and Affiliations

Hubei Provincial Clinical Research Center of Central Nervous System Repair and Functional Reconstruction, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei, 430000, China

湖北医科大学太和医院湖北省中枢神经系统修复与功能重建临床研究中心，湖北十堰，430000

Aixin Yu, Yiqi Wang, Chao Duan, Li Yu, Hui Gui, Jinxin Xin, Wendai Bao & Zhiqiang Dong

余爱新，王益琦，段超，李宇，惠贵，辛金新，鲍文岱，董志强

College of Biomedicine and Health, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, 430000, China

华中农业大学生命科学与技术学院生物医学与健康学院，湖北武汉，430000

Aixin Yu, Yiqi Wang, Chao Duan, Miaomiao Liao, Haiyan Zhu, Weiwei Tao, Wendai Bao & Zhiqiang Dong

余爱欣，王一琦，段超，廖妙淼，朱海燕，陶伟伟，鲍文岱，董志强

Department of Neurosurgery, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei, 430000, China

湖北医科大学太和医院神经外科，湖北十堰，430000

Jun Qin, Junrong Lei, Jun Liu & Zehao Yang

秦军、雷俊荣、刘军和杨泽浩

Central Laboratory, Hubei Cancer Hospital, Wuhan, Hubei, 430000, China

湖北省肿瘤医院中心实验室，湖北武汉，430000

Zhiqiang Dong

志江东

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Aixin Yu

余爱欣

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王益琦

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吴 Weiwei

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Contributions

捐款

Aixin Yu: Writing original draft, Validation, Methodology, Conception, Investigation. Yiqi Wang: Methodology, Investigation. Chao Duan: Methodology, Investigation. Jun Qin: Conception. Miaomiao Liao: Investigation. Haiyan Zhu: Investigation. Junrong Lei: Investigation. Jun Liu: Investigation. Zehao Yang: Investigation.

余爱欣：撰写原稿，验证，方法论，概念，调查。王一齐：方法论，调查。赵端：方法论，调查。秦军：构思。廖妙淼：调查。。雷俊荣：调查。刘军：调查。杨泽浩：调查。

Li Yu: Investigation. Hui Gui: Investigation. Jinxin Xin: Investigation. Weiwei Tao: Conception, Funding acquisition. Wendai Bao: Conception, Writing—review and editing, Supervision, Funding acquisition. Zhiqiang Dong: Conception, Writing—review and editing, Supervision, Funding acquisition. All authors: Final approval of manuscript..

。惠贵：调查。金欣欣：调查。陶伟伟：构思，资金获取。《文黛报》：构思、写作评论和编辑、监督、资金获取。董志强：构思，写作评论和编辑，监督，资金获取。所有作者：稿件的最终批准。。

Corresponding authors

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Correspondence to

通信对象

Wendai Bao

温台宝

或

Zhiqiang Dong

志江东

Ethics declarations

Competing interests

相互竞争的利益

The authors declare no competing interests.

作者声明没有利益冲突。

Ethics statement

道德声明

The animal study was performed after receiving approval from the Institutional Animal Care and Use Committee of Hubei University of Medicine (approval number: 2023008). Informed consents were obtained from GBM patients for sample collection, and the study was approved by the Ethics Committee of Shiyan Taihe Hospital (approval number: 2023KS11)..

动物研究是在获得湖北医科大学机构动物护理和使用委员会（批准号：2023008）批准后进行的。获得GBM患者的知情同意书进行样本采集，该研究得到十堰泰和医院伦理委员会的批准（批准号：2023KS11）。。

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Communications Biology

通信生物学

thanks Vivek Sharma and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Kaliya Georgieva. A peer review file is available.

感谢维韦克·夏尔马（VivekSharma）和另一位匿名审稿人对这项工作的同行评审做出的贡献。主要处理编辑：卡莉娅·乔治耶娃（KaliyaGeorgieva）。同行评审文件可用。

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Yu, A., Wang, Y., Duan, C.

于，A，王，Y，段，C。

et al.