USP15的N6甲基腺苷修饰通过AKT/mTOR信号激活途径抑制LGALS3泛素介导的降解来调节肝细胞癌的化疗耐药性-动脉网

N6-methyladenosine-modification of USP15 regulates chemotherapy resistance by inhibiting LGALS3 ubiquitin-mediated degradation via AKT/mTOR signaling activation pathway in hepatocellular carcinoma

Nature 等信源发布 2025-01-10 23:31

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可切换为仅中文

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Abstract

摘要

Hepatocellular carcinoma (HCC) is among the most malignant tumors and seriously threatens human health worldwide, and its incidence rate is increasing annually. USP15 is a member of the ubiquitination-specific protease (USP) family, which can regulate protein ubiquitination, thereby affecting their stability, and is dysregulated in many cancers, but its expression and regulatory mechanism in HCC are unclear.

肝细胞癌（HCC）是全球最恶性的肿瘤之一，严重威胁人类健康，其发病率呈逐年上升趋势。USP15是泛素化特异性蛋白酶（USP）家族的成员，其可以调节蛋白质泛素化，从而影响其稳定性，并且在许多癌症中失调，但其在HCC中的表达和调节机制尚不清楚。

The aims of this study were to explore the role and mechanism of USP15 in regulating HCC cell stemness, proliferation, and lenvatinib resistance. Immunohistochemistry and high-throughput sequencing analyses of tumor and adjacent normal tissue samples from 52 patients with HCC were conducted. Functional analyses of immortalized human liver and HCC cell lines were conducted, including quantitative real-time PCR; western blot; plasmid, lentivirus, and siRNA transfection; co-immunoprecipitation; mass spectrometry; MeRIP-qPCR; and ubiquitination, cell growth, colony formation, and spheroid formation assays.

本研究的目的是探讨USP15在调节HCC细胞干性，增殖和lenvatinib耐药性中的作用和机制。对52例HCC患者的肿瘤和邻近正常组织样本进行了免疫组织化学和高通量测序分析。进行了永生化人肝脏和HCC细胞系的功能分析，包括定量实时PCR；蛋白质印迹；质粒，慢病毒和siRNA转染；免疫共沉淀；质谱法；MeRIP qPCR；和泛素化，细胞生长，集落形成和球体形成测定。

HCC tumor growth was also assessed using cell transplantation in nude mice. We found that USP15 is upregulated in HCC and affects patient prognosis. Our results demonstrated that USP15 can increase LGALS3 stability in HCC through deubiquitination modification, and affect the stemness, proliferation, and lenvatinib resistance of HCC cells by activating the AKT/mTOR pathway.

还使用裸鼠中的细胞移植评估HCC肿瘤生长。我们发现USP15在HCC中上调并影响患者预后。我们的研究结果表明，USP15可以通过去泛素化修饰增加HCC中LGALS3的稳定性，并通过激活AKT/mTOR途径影响HCC细胞的干性，增殖和lenvatinib抗性。

USP15 expression levels were positively correlated with HCC cell stemness, proliferation, and lenvatinib resistance. In addition, methyltransferase-like protein 3 (Mettl3) N6-methyladenosine (m6A) modified USP15 to upregulate its levels by increasing its mRNA stability. These findings provide a theoretical basis for the potential discovery of new HCC oncogenes, as well as the identification of effec.

USP15表达水平与HCC细胞干性，增殖和lenvatinib耐药性呈正相关。此外，甲基转移酶样蛋白3（Mettl3）N6-甲基腺苷（m6A）修饰USP15，通过增加其mRNA稳定性来上调其水平。这些发现为新的HCC癌基因的潜在发现以及有效的鉴定提供了理论基础。

Background

背景

HCC is one of the most common malignant tumors and its incidence continues to increase worldwide. HCC is extremely malignant, with a 5-year overall survival rate (OS) of just 18%. Its onset is insidious, and by the time of diagnosis, approximately 80% of HCC patients have already reached an advanced stage, at which point surgery is no longer feasible [.

HCC是最常见的恶性肿瘤之一，其发病率在全球范围内持续增加。HCC恶性程度极高，5年总生存率（OS）仅为18%。。

Ubiquitination is a reversible post-translational modification that regulates protein degradation and signal transduction by covalently binding small molecules of ubiquitin to target proteins. The process of ubiquitination leads to protein degradation through ubiquitin-proteasomes and selective autophagy [.

泛素化是一种可逆的翻译后修饰，通过将泛素小分子与靶蛋白共价结合来调节蛋白质降解和信号转导。泛素化过程通过泛素蛋白酶体和选择性自噬导致蛋白质降解[。

], and is involved in many cellular life activities, such as cell cycle regulation, DNA repair, and signal transduction. Ubiquitination is also involved in regulating tumor cell proliferation, metastasis, and angiogenesis [

]，并参与许多细胞生命活动，例如细胞周期调控，DNA修复和信号转导。泛素化也参与调节肿瘤细胞的增殖、转移和血管生成[

]. Deubiquitinases can regulate the ubiquitination modification of proteins, thereby affecting their stability. The USP family, which includes USP15, constitutes the largest subfamily of deubiquitinases.

]。去泛素化酶可以调节蛋白质的泛素化修饰，从而影响其稳定性。USP家族（包括USP15）构成了去泛素化酶的最大亚家族。

USP15 is involved in various cell biological processes, such as proliferation, invasion, apoptosis, and transcriptional regulation, among others. Further, USP15 is upregulated in several malignancies including breast, ovarian, gastric, and bladder cancers, but downregulated in around 25% of pancreatic cancer and 11% of glioblastoma, indicating that USP15 potentially has dual roles in promoting and suppressing tumors [.

。此外，USP15在包括乳腺癌，卵巢癌，胃癌和膀胱癌在内的几种恶性肿瘤中上调，但在约25%的胰腺癌和11%的胶质母细胞瘤中下调，表明USP15可能在促进和抑制肿瘤中具有双重作用[。

It has been reported in that USP15 is highly expressed in gastric cancer, promoting malignant progression by activating the Wnt/β-catenin pathway [

据报道，USP15在胃癌中高表达，通过激活Wnt/β-连环蛋白途径促进恶性进展[

], however, other studies have found that USP15 can inhibit the NF-κB pathway by deubiquitinating IκB-α, thereby inhibiting the proliferation and invasion of gastric cancer [

]然而，其他研究发现USP15可以通过去泛素化IκB-α来抑制NF-κB通路，从而抑制胃癌的增殖和侵袭[

]. In the context of HCC, some studies have shown that USP15 is highly expressed in tumor tissues, promoting HCC cell proliferation and inhibiting apoptosis [

]。在HCC的背景下，一些研究表明USP15在肿瘤组织中高表达，促进HCC细胞增殖并抑制细胞凋亡[

]. However, another investigation found that USP15 increases KEAP1 stability through deubiquitination, thereby promoting Nrf2 degradation, which results in reactive oxygen species accumulation, ultimately inducing HCC cell apoptosis and inhibiting HCC proliferation [

]。然而，另一项研究发现，USP15通过去泛素化增加KEAP1的稳定性，从而促进Nrf2降解，从而导致活性氧的积累，最终诱导HCC细胞凋亡并抑制HCC增殖[

]. These studies demonstrate that the biological function of USP15 in cancers is complex and even contradictory. Hence, in-depth understanding of the role and mechanism underlying USP15 activity in HCC will be conducive to determining the processes involved in HCC occurrence and development, which are of great significance for the treatment of this type of cancer..

]。这些研究表明，USP15在癌症中的生物学功能是复杂的，甚至是矛盾的。因此，深入了解USP15活性在HCC中的作用和机制将有助于确定HCC发生和发展的过程，这对治疗此类癌症具有重要意义。。

In this study, we found that USP15 is highly expressed in HCC and affects patient prognosis. Further, our data show that USP15 can increase LGALS3 stability through deubiquitination modification, promote HCC cell stemness and proliferation by activating the AKT/mTOR pathway, and improve HCC resistance to lenvatinib.

在这项研究中，我们发现USP15在HCC中高度表达并影响患者预后。此外，我们的数据显示USP15可以通过去泛素化修饰增加LGALS3的稳定性，通过激活AKT/mTOR途径促进HCC细胞的干性和增殖，并改善HCC对lenvatinib的耐药性。

In addition, our results indicate that m6A modification by Mettl3 maintains high USP15 expression by increasing USP15 mRNA stability..

此外，我们的结果表明，Mettl3对m6A的修饰通过增加USP15 mRNA的稳定性来维持USP15的高表达。。

Results

结果

Expression characteristics and clinical significance of USP15 in HCC

USP15在肝癌中的表达特点及临床意义

We collected specimens from 52 patients with HCC who were treated at the Second Hospital of Jilin University from January 2019 to January 2021. Through high-throughput sequencing, we found that the

我们收集了2019年1月至2021年1月在吉林大学第二医院接受治疗的52例HCC患者的标本。通过高通量测序，我们发现

USP15

美国药典15

expression was higher in cancer tissue than that in paired adjacent tissues, suggesting that USP15 may affect HCC malignant progression (Fig.

癌组织中的表达高于配对的邻近组织，表明USP15可能影响HCC的恶性进展（图）。

1A, B

1A、B

). Subsequently, we performed qRT-PCR, WB, and immunohistochemistry analyses of the specimens from the 52 patients with HCC finding that USP15 expression levels in cancer tissues were significantly higher than those in adjacent tissues (Fig.

)。。

1C–E

). In addition, analysis of patient data indicated that USP15 expression level was positively correlated with tumor TNM stage, tumor size, cirrhosis, and microvascular invasion, while negatively correlated with patient overall survival (Supplementary Table

)。此外，对患者数据的分析表明，USP15表达水平与肿瘤TNM分期、肿瘤大小、肝硬化和微血管浸润呈正相关，而与患者总生存率呈负相关（补充表）

S2级

; Fig.

；图。

1层

). Data analysis from the GEPIA database (

)。来自GEPIA数据库的数据分析(

GEPIA (cancer-pku.cn)

GEPIA（北京大学癌症研究所）

)also showed that USP15 expression level was negatively associated with both overall survival and disease-free survival in patients with HCC (Fig.

)还显示USP15表达水平与HCC患者的总生存率和无病生存率呈负相关（图）。

1克

; Supplementary Fig.

；补充图。

S1C

S1C级

Fig. 1: Expression characteristics and clinical significance of USP15 in HCC.

图1:USP15在HCC中的表达特征和临床意义。

B类

Heat maps and volcano plots of the results of high-throughput sequencing of patient tissue specimens showing the difference in

患者组织标本高通量测序结果的热图和火山图显示了

USP15

美国药典15

expression between tumor and adjacent normal tissues(

肿瘤与邻近正常组织之间的表达(

Green represents tumor tissues, and red represents para - cancerous tissues;

绿色代表肿瘤组织，红色代表癌旁组织；

B类

Blue indicates high - expression, and red indicates low - expression).

蓝色表示高表达，红色表示低表达）。

C级

Comparison of USP15 expression in tumor and paired adjacent normal tissues from 52 patients with HCC detected by qRT-PCR and WB (52 specimens were examined, since 8 of them are the most representative and sufficient to stand for all 52 specimens, we did not insert all the specimens into the figure).

通过qRT-PCR和WB检测52例HCC患者的肿瘤和配对邻近正常组织中USP15表达的比较（检查了52例标本，因为其中8例最具代表性，足以代表所有52例标本，我们没有将所有标本插入图中）。

Comparison of USP15 expression in tumor and paired normal adjacent tissues from patients with HCC detected by immunohistochemistry(Detected at a magnification of 200 times with a scale bar of 60μm).

通过免疫组织化学检测来自HCC患者的肿瘤和配对正常邻近组织中USP15表达的比较（放大200倍，比例尺为60μm）。

F级

Kaplan-Meier analysis of the relationship between USP15 expression levels and prognosis in 52 patients with HCC.

Kaplan-Meier分析52例HCC患者USP15表达水平与预后的关系。

克

Analysis of the relationship between USP15 expression and prognosis of patients with HCC using data from the GEPIA database. Data are expressed as mean values,

使用GEPIA数据库的数据分析USP15表达与HCC患者预后之间的关系。数据表示为平均值，

< 0.05,

**p

< 0.01.

Full size image

全尺寸图像

Effect of USP15 on HCC cell stemness

USP15对肝癌细胞干性的影响

Our data suggest that USP15 may promote HCC malignant progression, though the specific function of USP15 in HCC remains unknown. First, the experimental cell lines Huh-7 (high USP15 expression), HCC-LM3 (high USP15 expression) and Hep-3B (relatively low USP15 expression) were screened through qRT-PCR and WB experiments, and stable knockdown of USP15 conducted by lentiviral transfection.

我们的数据表明USP15可能促进HCC恶性进展，尽管USP15在HCC中的具体功能仍然未知。首先，通过qRT-PCR和WB实验筛选实验细胞系Huh-7（高USP15表达），HCC-LM3（高USP15表达）和Hep-3B（相对低USP15表达），并通过慢病毒转染进行USP15的稳定敲低。

The resulting cell lines were the USP15-knockdown Huh-7, HCC-LM3, and the USP15-overexpressing cell line, Hep-3B (Supplementary Figs. .

产生的细胞系是USP15敲低的Huh-7，HCC-LM3和USP15过表达的细胞系Hep-3B（补充图）。

S1级

A,B;

A、 B；

S2A, B

S2A、B

). Next, we conducted qRT-PCR and WB experiments, which demonstrated positive correlations between levels of USP15 and those of molecules related to tumor stemness (CD133, EPCAM, SOX2, ALDH1 and OCT4) [

)。接下来，我们进行了定量rt-PCR和WB实验，结果表明USP15水平与肿瘤干细胞相关分子（CD133、EPCAM、SOX2、ALDH1和OCT4）水平呈正相关[

] (Fig.

]（图。

2A–D

). Further, in sphere-forming experiments, the knockdown of USP15 significantly reduced the sphere-forming ability of Huh-7 and HCC-LM3 cells, while that of Hep-3B cells overexpressing USP15 was enhanced (Fig.

)。此外，在球形成实验中，USP15的敲低显着降低了Huh-7和HCC-LM3细胞的球形成能力，而过表达USP15的Hep-3B细胞的球形成能力增强（图）。

2E, F

), indicating that USP15 promotes HCC cell stemness.

)，表明USP15促进HCC细胞干性。

Fig. 2: Effect of USP15 on HCC cell stemness.

图2:USP15对HCC细胞干性的影响。

A、，

B类

qRT-PCR analysis of expression of the tumor stemness-related genes,

肿瘤干性相关基因表达的qRT-PCR分析，

CD133

EPCAM

SOX2

SOX2公司

ALDH1

and

和

OCT4

10月4日

, in Huh7 and HCC-LM3 cells with USP15 knocked down and Hep3B cells overexpressing USP15.

，在USP15敲低的Huh7和HCC-LM3细胞中以及过表达USP15的Hep3B细胞中。

C、，

WB analysis of expression of the tumor stemness-related proteins, CD133, EPCAM, SOX2, ALDH1 and OCT4 in Huh7 and HCC-LM3 cells with USP15 knocked down and Hep3B cells overexpressing USP15.

WB分析USP15敲低的Huh7和HCC-LM3细胞和过表达USP15的Hep3B细胞中肿瘤干细胞相关蛋白CD133，EPCAM，SOX2，ALDH1和OCT4的表达。

E、，

F级

Sphere-formation assay to assess the primary and secondary sphere formation abilities of Huh7 and HCC-LM3 cells with USP15 knocked down and Hep3B cells overexpressing USP15. Images show secondary spheres formed by these cells. Data are expressed as mean values,

球形成测定以评估USP15敲低的Huh7和HCC-LM3细胞和过表达USP15的Hep3B细胞的初级和次级球形成能力。图像显示了由这些细胞形成的次级球体。数据表示为平均值，

< 0.05,

**p

< 0.01.

Full size image

全尺寸图像

Effect of USP15 on HCC proliferation and lenvatinib resistance

Several studies have shown that tumor drug resistance is significantly related to their stemness properties [

多项研究表明，肿瘤耐药性与其干性有显著关系[

]. Therefore, we examined whether USP15 influences HCC resistance to lenvatinib. In colony formation experiments, the same concentration of lenvatinib was added to each experimental group, and the colony formation rate of Huh-7 and HCC-LM3 cells with USP15 knocked down was significantly lower than that of corresponding wild-type HCC cells, DMSO was added to cells with USP15 knocked down as a control group.

]。因此，我们检查了USP15是否影响HCC对lenvatinib的耐药性。在集落形成实验中，每个实验组加入相同浓度的lenvatinib，USP15敲除的Huh-7和HCC-LM3细胞的集落形成率显着低于相应的野生型HCC细胞，DMSO被添加到USP15敲除的细胞中作为对照组。

The colony formation rates of Huh-7 and HCC-LM3 cells were also significantly lower than those of wild-type HCC cells. In Hep-3B overexpressing USP15, the opposite results were observed (Fig. .

Huh-7和HCC-LM3细胞的集落形成率也显着低于野生型HCC细胞。在过表达USP15的Hep-3B中，观察到相反的结果（图）。

3A, B

3A，B

). Additionally, a CCK8 experiment conducted by us showed that, over time, the viability of USP15-knockdown Huh-7 and HCC-LM3 cells treated with the same concentration of lenvatinib was lower than that of wild-type HCC cells. Conversely, in USP15-overexpressing Hep-3B cells, the opposite results were observed (Fig.

)。此外，我们进行的CCK8实验表明，随着时间的推移，用相同浓度的lenvatinib处理的USP15敲低Huh-7和HCC-LM3细胞的活力低于野生型HCC细胞。相反，在USP15过表达Hep-3B细胞中，观察到相反的结果（图）。

3C, D

3C，D

). These findings demonstrate that USP15 promotes both HCC cell proliferation and increases resistance to lenvatinib in HCC cells.

)。这些发现表明USP15促进HCC细胞增殖并增加HCC细胞对lenvatinib的抗性。

Fig. 3: Effects of USP15 on HCC cell proliferation and lenvatinib resistance.

图3:USP15对HCC细胞增殖和lenvatinib耐药性的影响。

A、，

B类

Colony formation assay to analyze the colony formation rate of Huh7 and HCC-LM3 cells with USP15 knocked down (

集落形成试验分析USP15敲除后Huh7和HCC-LM3细胞的集落形成率(

) and Hep3B cells overexpressing USP15 (

)和过表达USP15的Hep3B细胞(

B类

) following treatment with lenvatinib.

)用lenvatinib治疗后。

C、，

CCK8 assay to analyze the survival rate of Huh7 and HCC-LM3 cells with USP15 knocked down (

CCK8法分析USP15敲除后Huh7和HCC-LM3细胞的存活率(

C级

) and Hep3B cells overexpressing USP15 (

)和过表达USP15的Hep3B细胞(

) following treatment with lenvatinib. Data are expressed as mean values,

)用lenvatinib治疗后。数据表示为平均值，

< 0.05,

**p

< 0.01.

Full size image

全尺寸图像

In vivo experiments

体内实验

To further verify the above results, we conducted a tumor formation experiment in nude mice. Tumors transplanted in nude mice inoculated with Huh7-shUSP15 were significantly smaller than those in mice inoculated with wild-type Huh7 cells. Additionally, when nude mice in both the experimental and control groups received 50 mg/kg lenvatinib injections via the tail vein, the transplanted tumors in mice inoculated with Huh7-shUSP15 cells were smaller than those in mice inoculated with wild-type Huh7 cells (Fig.

为了进一步验证上述结果，我们在裸鼠中进行了肿瘤形成实验。接种Huh7-shUS15的裸鼠移植的肿瘤明显小于接种野生型Huh7细胞的小鼠。此外，当实验组和对照组的裸鼠通过尾静脉接受50mg/kg lenvatinib注射时，接种Huh7-shUS15细胞的小鼠的移植肿瘤小于接种野生型Huh7细胞的小鼠（图1）。

4A–C

). Immunohistochemistry analysis of tumor tissue samples from mice in each group was also conducted to assess USP15 expression levels (Fig.

)。还对每组小鼠的肿瘤组织样品进行了免疫组织化学分析，以评估USP15的表达水平（图）。

4D级

Fig. 4: USP15 promotes HCC proliferation and improves lenvatinib resistance in vivo.

图4:USP15促进HCC增殖并改善体内lenvatinib抗性。

Nude mouse tumor formation experiment. Compared with mice injected with wild-type Huh7 cells, those inoculated with Huh7 cells with USP15 knocked down generated significantly smaller subcutaneous tumors. After injection of mice with lenvatinib via the tail vein, mice inoculated with Huh7 cells with USP15 knocked down had smaller subcutaneous tumors than those in mice inoculated with wild-type Huh7 cells.

裸鼠肿瘤形成实验。与注射野生型Huh7细胞的小鼠相比，用USP15敲低的Huh7细胞接种的小鼠产生的皮下肿瘤明显更小。通过尾静脉注射lenvatinib后，接种USP15敲除的Huh7细胞的小鼠的皮下肿瘤比接种野生型Huh7细胞的小鼠的皮下肿瘤小。

B类

Subcutaneous transplanted tumor volume in nude mice of each group.

各组裸鼠皮下移植瘤体积。

C级

Subcutaneous transplanted tumor mass in each group of nude mice.

每组裸鼠皮下移植瘤块。

Immunohistochemical analysis of the expression of USP15 in subcutaneous transplanted tumor specimens of four groups of nude mice (Detected at a magnification of 200 times with a scale bar of 60μm). Data are expressed as mean values,

免疫组织化学分析USP15在四组裸鼠皮下移植肿瘤标本中的表达（放大200倍，比例尺为60μm）。数据表示为平均值，

< 0.05,

**p

< 0.01.

Full size image

全尺寸图像

Overall, our data indicate that USP15 is highly expressed in HCC and is negatively correlated with patient prognosis. Our findings also indicate that USP15 promotes HCC cell stemness and proliferation, as well as enhancing resistance to lenvatinib in HCC.

总体而言，我们的数据表明USP15在HCC中高度表达，并且与患者预后呈负相关。我们的研究结果还表明USP15促进HCC细胞的干性和增殖，并增强HCC对lenvatinib的耐药性。

LGALS3 is downstream of USP15 and LGALS3 interacts with USP15 to promote HCC stemness, proliferation, and lenvatinib resistance through AKT/m-TOR activation

LGALS3位于USP15的下游，LGALS3与USP15相互作用，通过AKT/m-TOR激活促进HCC干细胞、增殖和lenvatinib耐药

In previous experiments, we found that USP15 can promote HCC cell stemness and proliferation and increase HCC resistance to lenvatinib, but the specific mechanism involved remains to be elucidated. Therefore, we conducted high-throughput sequencing of samples from HCC cells that expressed different levels of USP15 to identify differentially expressed genes, followed by KEGG pathway enrichment analysis.

在以前的实验中，我们发现USP15可以促进HCC细胞的干性和增殖，并增加HCC对lenvatinib的耐药性，但所涉及的具体机制仍有待阐明。因此，我们对表达不同水平USP15的HCC细胞样品进行了高通量测序，以鉴定差异表达的基因，然后进行KEGG途径富集分析。

The results indicated that USP15 may act through the AKT/m-TOR pathway (Fig. .

结果表明USP15可能通过AKT/m-TOR途径起作用（图）。

5A–C

Fig. 5: LGALS3 is located downstream of USP15 and its protein expression is regulated by USP15.

图5:LGALS3位于USP15的下游，其蛋白质表达受USP15调节。

A, B

A、 B类

High-throughput sequencing analysis of HCC cells differentially expressing USP15; heat maps and volcano maps showing differentially expressed genes (Blue indicates high - expression, and red indicates low - expression).

差异表达USP15的HCC细胞的高通量测序分析；热图和火山图显示差异表达的基因（蓝色表示高表达，红色表示低表达）。

C级

KEGG analysis of pathways enriched for differentially expressed genes.

KEGG分析富含差异表达基因的途径。

Mass spectrometry analysis identifying LGALS3 as a molecule co-expressed with USP15.

质谱分析将LGALS3鉴定为与USP15共表达的分子。

Immunoprecipitation experiments verifying that USP15 and LGALS3 can physically interact.

免疫沉淀实验验证USP15和LGALS3可以物理相互作用。

F, G

F、克

WB and qRT-PCR experiments to analyze the relationship between USP15 and LGALS3. USP15 and LGALS3 were positively correlated at the protein, but not at the mRNA, level. Data are expressed as mean values,

WB和qRT-PCR实验分析USP15和LGALS3之间的关系。USP15和LGALS3在蛋白质水平上呈正相关，但在mRNA水平上不相关。数据表示为平均值，

< 0.05,

**p

< 0.01.

Full size image

全尺寸图像

To explore proteins that interact with USP15, we next performed mass spectrometry analysis on transfected HCC cells and identified LGALS3 as a molecule co-expressed with USP15. We then performed Co-IP experiments and found that USP15 and LGALS3 could physically interact (Fig.

为了探索与USP15相互作用的蛋白质，我们接下来对转染的HCC细胞进行了质谱分析，并将LGALS3鉴定为与USP15共表达的分子。然后，我们进行了Co-IP实验，发现USP15和LGALS3可以物理相互作用（图）。

5D, E

5D，E

). To further explore the relationship between these two molecules, we performed qRT-PCR and WB experiments finding that LGALS3 is downstream of USP15. Additionally, our data indicate that USP15 can regulate LGALS3 at the protein level, while knockdown or overexpression of

)。为了进一步探索这两个分子之间的关系，我们进行了qRT-PCR和WB实验，发现LGALS3位于USP15的下游。此外，我们的数据表明USP15可以在蛋白质水平上调节LGALS3，同时敲低或过表达

USP15

美国药典15

at the mRNA level does not affect on LGALS3 (Fig.

在mRNA水平上不影响LGALS3（图）。

5F, G

5F，G

LGALS3, a member of the galectin family, has been reported to be closely associated with tumor occurrence and development [

LGALS3是半乳糖凝集素家族的成员，据报道与肿瘤的发生和发展密切相关[

]. To investigate the biological role of LGALS3 in HCC, we repeated our previous experiments by performing qRT-PCR on 52 HCC tissue specimens. We found that LGALS3 was highly expressed in HCC tissues (Fig.

]。为了研究LGALS3在HCC中的生物学作用，我们通过对52个HCC组织标本进行qRT-PCR重复了我们之前的实验。我们发现LGALS3在HCC组织中高表达（图）。

), indicating that it may play an oncogenic role. In subsequent experiments, we used siRNA and transfection to construct lines with LGALS3 overexpressed and knocked down in Huh-7 and Hep-3B cells stably transfected with USP15. We further performed qRT-PCR, spheroid formation, and CCK8 assays on these cell lines and found that, similar to USP15, LGALS3 promotes HCC cell stemness and proliferation and enhances their resistance to lenvatinib (Fig.

)，表明它可能起致癌作用。在随后的实验中，我们使用siRNA和转染构建了在USP15稳定转染的Huh-7和Hep-3B细胞中过表达和敲除LGALS3的细胞系。我们进一步对这些细胞系进行了qRT-PCR，球体形成和CCK8测定，发现与USP15相似，LGALS3促进HCC细胞的干性和增殖，并增强其对lenvatinib的抗性（图）。

6B–G

Fig. 6: LGALS3 interacts with USP15 to promote HCC stemness, proliferation, and lenvatinib resistance through AKT/m-TOR activation.

图6:LGALS3与USP15相互作用，通过AKT/m-TOR激活促进HCC干细胞，增殖和lenvatinib抗性。

qRT-PCR analysis of

qRT-PCR分析

LGALS3

expression in tumor and paired adjacent tissues from 52 patients with HCC.

。

B, C

B、 C级

qRT-PCR analysis of the expression of the tumor stemness-related genes

肿瘤干细胞相关基因表达的qRT-PCR分析

CD133

EPCAM

SOX2

SOX2公司

ALDH1

and

和

OCT4

10月4日

in Huh7 cells with USP15 knocked down and LGALS3 overexpressed, and in Hep3B cells with USP15 overexpressed and LGALS3 knocked down.

在USP15敲低且LGALS3过表达的Huh7细胞中，以及在USP15过表达且LGALS3敲低的Hep3B细胞中。

D、，

Analysis of the primary and secondary sphere formation abilities of Huh7 cells with USP15 knocked down and LGALS3 overexpressed, and of Hep3B cells with USP15 overexpressed and LGALS3 knocked down.

分析USP15敲低和LGALS3过表达的Huh7细胞以及USP15过表达和LGALS3敲低的Hep3B细胞的一级和二级球体形成能力。

F、，

克

CCK8 assay to analyze survival rates following treatment with lenvatinib of Huh7 cells with USP15 knocked down and LGALS3 overexpressed and of Hep3B cells with USP15 overexpressed and LGALS3 knocked down.

CCK8分析用于分析lenvatinib处理USP15敲低和LGALS3过表达的Huh7细胞以及USP15过表达和LGALS3敲低的Hep3B细胞后的存活率。

小时，

我

WB assay of AKT/m-TOR pathway-related protein expression in Huh7 and Hep3B cells differentially expressing USP15 and LGALS3. Data are expressed as mean values,

WB测定差异表达USP15和LGALS3的Huh7和Hep3B细胞中AKT/m-TOR途径相关蛋白的表达。数据表示为平均值，

< 0.05,

**p

< 0.01.

Full size image

全尺寸图像

Therefore, we hypothesize that USP15 and LGALS3 act together to influence HCC malignant progression, though the pathways through which they operate remain to be verified. We subsequently performed WB on HCC cell lines with differential USP15 and LGALS3 expression to assess the levels of proteins associated with the AKT/mTOR pathway.

因此，我们假设USP15和LGALS3共同作用以影响HCC恶性进展，尽管它们的作用途径仍有待验证。随后，我们对具有差异USP15和LGALS3表达的HCC细胞系进行了WB，以评估与AKT/mTOR途径相关的蛋白质水平。

The results showed that USP15 and LGALS3 act together to increase the expression levels of p-AKT and p-mTOR. This activation of the AKT/mTOR pathway promotes HCC cell stemness and proliferation and enhances resistance to lenvatinib (Fig. .

结果显示USP15和LGALS3共同作用以增加p-AKT和p-mTOR的表达水平。AKT/mTOR途径的这种激活促进了HCC细胞的干性和增殖，并增强了对lenvatinib的抗性（图）。

6H, I

6小时，我

USP15 mediates LGALS3 expression through deubiquitination modification

USP15通过去泛素化修饰介导LGALS3的表达

In previous experiments, we found that USP15 and LGALS3 can interact, and that both are up-regulated in HCC. To explore whether there is a correlation between the high expression levels of USP15 and LGALS3, we conducted a protein half-life experiment. The results showed that overexpression of USP15 significantly increased the stability of LGALS3 (Fig.

在以前的实验中，我们发现USP15和LGALS3可以相互作用，并且两者在HCC中都被上调。为了探索USP15和LGALS3的高表达水平之间是否存在相关性，我们进行了蛋白质半衰期实验。结果表明，USP15的过表达显着增加了LGALS3的稳定性（图）。

7A个

). Given that USP15 is a deubiquitinating enzyme, we hypothesized that USP15 may improve LGALS3 stability by inhibiting its ubiquitination and degradation. To test this hypothesis, we conducted ubiquitination experiments and found that USP15 overexpression inhibited the LGALS3 ubiquitination-mediated degradation, proving that USP15 affects LGALS3 stability through deubiquitination modification (Fig.

)。鉴于USP15是一种去泛素化酶，我们假设USP15可能通过抑制其泛素化和降解来提高LGALS3的稳定性。为了验证这一假设，我们进行了泛素化实验，发现USP15过表达抑制了LGALS3泛素化介导的降解，证明USP15通过去泛素化修饰影响LGALS3的稳定性（图）。

7B, C

7B，C

). This finding also clarifies our data showing that USP15 regulates LGALS3 at the protein level without affecting its mRNA level.

)。这一发现也澄清了我们的数据，表明USP15在蛋白质水平上调节LGALS3而不影响其mRNA水平。

Fig. 7: USP15 mediates LGALS3 expression through deubiquitination modification.

图7:USP15通过去泛素化修饰介导LGALS3表达。

Protein half-life assay to assess LGALS3 stability in wild-type and USP15-overexpressing Hep3B cells. LGALS3 protein stability was increased in Hep3B cells overexpressing USP15.

蛋白质半衰期测定，以评估野生型和USP15过表达Hep3B细胞中LGALS3的稳定性。过表达USP15的Hep3B细胞中LGALS3蛋白的稳定性增加。

B、，

C级

Ubiquitination assay to analyze the effect of USP15 on LGALS3 ubiquitination-mediated degradation. LGALS3 ubiquitination-mediated degradation was significantly reduced following USP15 overexpression.

泛素化测定以分析USP15对LGALS3泛素化介导的降解的影响。USP15过表达后，LGALS3泛素化介导的降解显着降低。

Full size image

全尺寸图像

m6A modification mediates USP15 expression upregulation in HCC

m6A修饰介导HCC中USP15表达上调

Analysis using RMBase (

使用RMBase进行分析(

https://rna.sysu.edu.cn/rmbase/m6Amod.php

) indicated that

)表明

USP15

美国药典15

contains numerous m6A binding sites. Therefore, we hypothesize that high USP15 expression in HCC is related to its m6A modification, which is the most common and abundant post-transcriptional modification in eukaryotic RNA. Mettl3 is a methyltransferase that is key to regulation of m6A modification.

包含许多m6A结合位点。因此，我们假设USP15在HCC中的高表达与其m6A修饰有关，m6A修饰是真核RNA中最常见和最丰富的转录后修饰。Mettl3是一种甲基转移酶，是调节m6A修饰的关键。

Mettl3 expression is upregulated in HCC, but its specific functional mechanism in this context is unclear [.

Mettl3表达在HCC中上调，但其在这种情况下的具体功能机制尚不清楚[。

First, we conducted MeRIP-qPCR experiments, which demonstrated significantly higher

首先，我们进行了MeRIP qPCR实验，结果表明

USP15

美国药典15

m6A enrichment levels in the Huh-7 and HCC-LM3 cell lines than that in the immortalized human liver cell line, LO2. Next, we performed qRT-PCR analysis on samples from the 52 pairs of HCC tissue samples described above finding that

Huh-7和HCC-LM3细胞系中的m6A富集水平比永生化人肝细胞系LO2中的m6A富集水平高。接下来，我们对上述52对HCC组织样本的样本进行了qRT-PCR分析，发现

Mettl3

levels were significantly higher in HCC tumor specimens than those in para-cancerous tissues, analysis of GEPIA database data further verified this result (Fig.

HCC肿瘤标本中的水平显着高于癌旁组织，GEPIA数据库数据的分析进一步证实了这一结果（图）。

8A–C

Fig. 8: m6A modification mediates USP15 expression upregulation in HCC.

图8:m6A修饰介导HCC中USP15表达上调。

MeRIP-qPCR assay to detect m6A enrichment in Huh7 and HCC-LM3 cells.

MeRIP qPCR测定法检测Huh7和HCC-LM3细胞中m6A的富集。

B类

qRT-PCR assay to detect

qRT-PCR检测

Mettl3

expression levels in tumor and paired adjacent normal tissues from 52 patients with HCC.

52例HCC患者肿瘤和配对邻近正常组织中的表达水平。

C级

Analysis of data from the GEPIA database showing that Mettl3 expression level was positively correlated with that of USP15.

对GEPIA数据库数据的分析表明，Mettl3表达水平与USP15呈正相关。

D、，

qRT-PCR analysis of the effect of Mettl3 knockdown or overexpression on

qRT-PCR分析Mettl3敲低或过表达对

USP15

美国药典15

expression.

表达式。

F级

WB assay to analyze the effect of Mettl3 knockdown or overexpression on USP15 expression.

WB分析以分析Mettl3敲低或过表达对USP15表达的影响。

克

小时

MeRIP-qPCR assay to analyze the effect of Mettl3 knockdown or overexpression on m6A enrichment in the

MeRIP qPCR分析Mettl3敲低或过表达对m6A富集的影响

USP15

美国药典15

gene.

基因。

我

Actinomycin D assay to determine the effect of Mettl3 knockdown and overexpression on USP15 stability. Data are expressed as mean values,

放线菌素D测定以确定Mettl3敲低和过表达对USP15稳定性的影响。数据表示为平均值，

< 0.05,

**p

< 0.01.

Full size image

全尺寸图像

We then used siRNA and transfection to construct HCC cell lines with Mettl3 overexpressed and knocked down. Through qRT-PCR and WB experiments, we found that overexpression of Mettl3 significantly increased levels of USP15, while knocking down Mettl3 reduced USP15 expression (Fig.

然后，我们使用siRNA和转染构建Mettl3过表达和敲除的HCC细胞系。通过qRT-PCR和WB实验，我们发现Mettl3的过表达显着增加了USP15的水平，而敲低Mettl3降低了USP15的表达（图）。

8D–F

). MeRIP-qPCR experiments showed that m6A enrichment of

)。MeRIP qPCR实验表明，m6A富集

USP15

美国药典15

in HCC cells decreased after the knockdown of Mettl3, while Mettl3 overexpression significantly increased the m6A enrichment of

在敲除Mettl3后，HCC细胞中的表达减少，而Mettl3过表达显着增加了m6A的富集

USP15

美国药典15

. In an actinomycin D experiment, knockdown of Mettl3 reduced USP15 stability, while overexpressing Mettl3 increased USP15 stability (Fig.

在放线菌素D实验中，Mettl3的敲低降低了USP15的稳定性，而过表达Mettl3增加了USP15的稳定性（图）。

8G–I

). These data indicate that m6A modification mediates the high USP15 expression observed in HCC.

)。这些数据表明m6A修饰介导在HCC中观察到的高USP15表达。

Discussion

讨论

USP15 is a deubiquitinase that can increase protein stability through deubiquitination modification and participate in various intracellular processes, such as cell proliferation, apoptosis, autophagy, and immune responses [

USP15是一种去泛素化酶，可以通过去泛素化修饰增加蛋白质的稳定性，并参与各种细胞内过程，如细胞增殖、凋亡、自噬和免疫反应[

]. Moreover, USP15 is upregulated in many cancers and increases the stability of oncoproteins through deubiquitination modification, thereby promoting cancer progression [

]。此外，USP15在许多癌症中上调，并通过去泛素化修饰增加癌蛋白的稳定性，从而促进癌症进展[

]. However, in some tumors, there is evidence that USP15 has both pro-oncogenic and tumor suppressor roles [

]。然而，在某些肿瘤中，有证据表明USP15同时具有促癌和抑癌作用[

], indicating that the role of USP15 in cancer is complex and sometimes contradictory, and highlighting it as an important and promising target in tumor treatment. With the incidence and mortality of HCC are increasing annually, understanding the role and mechanisms of USP15 activity in HCC is of great significance..

]，表明USP15在癌症中的作用是复杂的，有时是矛盾的，并强调它是肿瘤治疗中重要且有希望的靶标。随着HCC的发病率和死亡率逐年增加，了解USP15活性在HCC中的作用和机制具有重要意义。。

The mechanism underlying HCC drug resistance is highly complex. This resistance may be related to the biological function of the liver, which can metabolize drugs, making it difficult for drugs to reach therapeutic concentrations in this organ. Drug resistance of HCC may also be related to increased expression of transporters, inactivation of signaling pathways that regulate apoptosis, redistribution of drugs in cells, and activation of tumor stem cells [.

HCC耐药的机制非常复杂。这种耐药性可能与肝脏的生物学功能有关，肝脏可以代谢药物，使药物难以在该器官中达到治疗浓度。HCC的耐药性也可能与转运蛋白表达增加，调节细胞凋亡的信号通路失活，药物在细胞中的重新分布以及肿瘤干细胞的激活有关。

]. In this study, we demonstrate the role of USP15 in HCC progression and lenvatinib resistance and, for the first time, confirm that Mettl3-mediated m6A modification is involved in USP15 upregulation in HCC. In summary, our findings demonstrate that USP15 is highly expressed in HCC, with levels positively correlated with serum AFP levels, tumor TNM stage, tumor size, cirrhosis, and microvascular invasion, and negatively correlated with patient overall survival.

]。在这项研究中，我们证明了USP15在HCC进展和lenvatinib耐药中的作用，并首次证实Mettl3介导的m6A修饰参与了HCC中USP15的上调。总之，我们的研究结果表明USP15在HCC中高度表达，其水平与血清AFP水平，肿瘤TNM分期，肿瘤大小，肝硬化和微血管浸润呈正相关，与患者总生存率呈负相关。

Both in vitro and in vivo experiments demonstrated that USP15 promotes HCC stemness, proliferation, and increases HCC resistance to lenvatinib..

体外和体内实验均表明USP15促进HCC干性，增殖，并增加HCC对lenvatinib的耐药性。。

LGALS3 is a member of the galectin family that is mainly located in the cell cytoplasm. The N-terminus of LGALS3 is sensitive to matrix metalloproteinases and can interact with some proteins to participate in cell proliferation, apoptosis, pre-mRNA splicing, angiogenesis, inflammation, and fibrosis [.

LGALS3是半乳糖凝集素家族的成员，主要位于细胞质中。LGALS3的N端对基质金属蛋白酶敏感，可以与某些蛋白质相互作用，参与细胞增殖，凋亡，前mRNA剪接，血管生成，炎症和纤维化[。

]. LGALS3 is a stable biomarker with elevated expression levels in many diseases, including heart, kidney, and liver disorders, as well as cancer [

]。LGALS3是一种稳定的生物标志物，在许多疾病中表达水平升高，包括心脏、肾脏和肝脏疾病以及癌症[

]. In tumors, LGALS3 is upregulated in glioblastoma and can increase its sensitivity to radiotherapy and chemotherapy [

]。在肿瘤中，LGALS3在胶质母细胞瘤中上调，可以增加其对放疗和化疗的敏感性[

]. In addition, LGALS3 plays significant roles in hepatitis, cirrhosis, and liver failure. It is also highly expressed in HCC, where it may have a pro-cancer function [

]。此外，LGALS3在肝炎，肝硬化和肝衰竭中起着重要作用。它在HCC中也高度表达，可能具有促癌功能[

]. In this study, we found that LGALS3 functions downstream of USP15 and is upregulated in HCC, where it can interact with USP15 to promote HCC cell stemness, proliferation, and lenvatinib resistance. Furthermore, our data demonstrate that USP15 enhances LGALS3 stability and maintains its upregulated expression through deubiquitination modification, potentially revealing the mechanism of action of LGALS3 in HCC(Supplementary Fig.

]。在这项研究中，我们发现LGALS3在USP15的下游起作用，并在HCC中上调，它可以与USP15相互作用以促进HCC细胞的干性，增殖和lenvatinib耐药性。此外，我们的数据表明USP15通过去泛素化修饰增强LGALS3的稳定性并维持其上调的表达，可能揭示LGALS3在HCC中的作用机制（Supplementary Fig.）。

S3级

The PI3K/AKT/mTOR pathway is a classical pathway that responds to insulin signaling and acts downstream of tyrosine kinase receptor to function in cell growth, migration, and survival. In HCC, approximately 5%–10% of patients have abnormal activation of the PI3K/AKT/mTOR pathway, and inhibiting this pathway can significantly inhibit HCC cell proliferation and invasion [.

PI3K/AKT/mTOR途径是一种经典途径，可响应胰岛素信号传导，并在酪氨酸激酶受体的下游起作用，从而在细胞生长，迁移和存活中发挥作用。在HCC中，大约5%–10%的患者PI3K/AKT/mTOR通路异常激活，抑制该通路可以显着抑制HCC细胞的增殖和侵袭[。

]. Moreover, previous studies have shown that USP15 promotes colon cancer proliferation and invasion via the AKT/mTOR pathway [

]。此外，先前的研究表明USP15通过AKT/mTOR途径促进结肠癌的增殖和侵袭[

]. In our study, we found that USP15 can interact with LGALS3 and activate the AKT/mTOR pathway by phosphorylating the AKT and mTOR proteins to exert cancer-promoting functions. However, the mechanism by which USP15 activates AKT/mTOR signaling remains unclear. TGF-β has been reported to promote

]。在我们的研究中，我们发现USP15可以通过磷酸化AKT和mTOR蛋白来发挥促癌功能，从而与LGALS3相互作用并激活AKT/mTOR途径。然而，USP15激活AKT/mTOR信号传导的机制仍不清楚。据报道，TGF-β可促进

USP15

美国药典15

mRNA translation through PI3K/AKT signaling, thereby activating the AKT/mTOR pathway [

通过PI3K/AKT信号传导进行mRNA翻译，从而激活AKT/mTOR途径[

RNA modification is a dynamic and reversible process, with m6A modification the most common of all RNA epigenetic alterations discovered to date. It regulates RNA transcription, editing, translation, and stability, playing a leading role in tumor cell proliferation and invasion [

RNA修饰是一个动态且可逆的过程，m6A修饰是迄今为止发现的所有RNA表观遗传改变中最常见的。它调节RNA的转录、编辑、翻译和稳定性，在肿瘤细胞的增殖和侵袭中起主导作用[

]. The up-regulation or down-regulation of m6A modification and its regulatory factors are critical in HCC occurrence and development [

]。m6A修饰及其调控因子的上调或下调在HCC的发生和发展中至关重要[

]. Mettl3 is upregulated in various HCC cell lines, resulting in increased m6A modification and promoting HCC progression. However, the mechanism of action of Mettl3 in the context of HCC remains unclear [

]。Mettl3在各种HCC细胞系中上调，导致m6A修饰增加并促进HCC进展。然而，Mettl3在肝癌中的作用机制仍不清楚[

]. Our study demonstrated that USP15 has numerous Mettl3 binding sites and that Mettl3 is highly expressed in HCC. Moreover, Mettl3 expression level directly influenced

]。我们的研究表明USP15具有许多Mettl3结合位点，并且Mettl3在HCC中高度表达。此外，Mettl3表达水平直接影响

USP15

美国药典15

m6A enrichment, indicating that USP15 expression upregulation in HCC is likely mediated by m6A modification. Subsequently, we found that Mettl3 expression level can affect

m6A富集，表明HCC中USP15表达上调可能是由m6A修饰介导的。随后，我们发现Mettl3表达水平可以影响

USP15

美国药典15

mRNA stability. Therefore, we conclude that USP15 upregulation in HCC is likely mediated through m6A modification.

mRNA稳定性。因此，我们得出结论，USP15在HCC中的上调可能是通过m6A修饰介导的。

Despite efforts to ensure a rigorous experimental design, our study had some limitations. Although we found that USP15 increased the resistance to lenvatinib in HCC, we did not study specimens from patients with HCC treated with lenvatinib, making it impossible for us to objectively evaluate whether USP15 can be used as a marker for targeted treatment resistance to lenvatinib in the clinic.

尽管努力确保严格的实验设计，但我们的研究仍有一些局限性。尽管我们发现USP15增加了HCC对lenvatinib的耐药性，但我们没有研究用lenvatinib治疗的HCC患者的标本，因此我们不可能客观评估USP15是否可以作为临床上对lenvatinib靶向治疗耐药性的标志物。

In addition, the specific mechanism underlying USP15-mediated activation of the AKT/mTOR pathway was not verified, and will be investigated in subsequent studies..

此外，USP15介导的AKT/mTOR途径激活的具体机制尚未得到验证，将在随后的研究中进行研究。。

Materials and methods

材料和方法

Patients and clinical specimens

患者和临床标本

Tumor and corresponding normal control tissue specimens were collected from 52 patients diagnosed with HCC who underwent liver resection at the Second Hospital of Jilin University from January 2019 to January 2021. Patient data were also collected, including sex, age, etiology, serum AFP, liver cirrhosis, tumor stage, tumor size, smoking history, alcohol consumption history, and microvascular invasion.

从2019年1月至2021年1月在吉林大学第二医院接受肝切除术的52例诊断为HCC的患者中收集肿瘤和相应的正常对照组织标本。。

Cancer staging was based on the HCC-TNM staging of the International Union Against Cancer. All human sample studies were reviewed and approved by the Ethics Committee of the Second Hospital of Jilin University, and informed consent was obtained from all patients. All procedures were in accordance with the Declaration of Helsinki..

癌症分期基于国际抗癌联盟的HCC-TNM分期。吉林大学第二医院伦理委员会对所有人体样本研究进行了审查和批准，并获得了所有患者的知情同意。所有程序均符合赫尔辛基宣言。。

Cell culture

细胞培养

The human liver immortalized cell line, LO2, and the HCC cell lines, Hep3B, SK-Hep1, Huh7, HCC-LM3, and SNU-449, were purchased from the American Type Bioresource Collection. Cells were cultured in appropriate complete medium (DMEM; BBI) containing 10% fetal bovine serum (BBI), 1% penicillin (100 U/ml), and streptomycin (100 U/ml).

人肝永生化细胞系LO2和HCC细胞系Hep3B，SK-Hep1，Huh7，HCC-LM3和SNU-449购自美国典型生物资源保藏中心。将细胞在含有10%胎牛血清（BBI），1%青霉素（100 U/ml）和链霉素（100 U/ml）的适当完全培养基（DMEM；BBI）中培养。

All cells were cultured in a humidified incubator at 37°C with 5% CO.

将所有细胞在37℃，5%CO的潮湿培养箱中培养。

Reagents and antibodies

试剂和抗体

Trypsin (0.25%), TRIzol, and RIPA lysis buffer were purchased from Sangon Biotech (Shanghai, China). Lipofectamine 3000 was from Invitrogen (Grand Island, NY, USA). An RNA Reverse Transcription Kit was purchased from Thermo (Waltham, Massachusetts, USA). Antibodies (anti-USP15, anti-LGALS3, anti-Mettl3, anti-AKT, anti-p-AKT, anti-mTOR, anti-p-mTOR, and anti-GAPDH) were all purchased from Proteintech (Rosemont, Illinois.

胰蛋白酶（0.25%），TRIzol和RIPA裂解缓冲液购自Sangon Biotech（中国上海）。。RNA逆转录试剂盒购自Thermo（Waltham，Massachusetts，USA）。抗体（抗USP15，抗LGALS3，抗Mettl3，抗AKT，抗p-AKT，抗mTOR，抗p-mTOR和抗GAPDH）均购自Proteintech（Rosemont，Illinois）。

USA). Unless otherwise stated, all other chemical reagents were purchased from Sigma-Aldrich (St. Louis, Missouri, USA)..

美国）。除非另有说明，否则所有其他化学试剂均购自Sigma-Aldrich（美国密苏里州圣路易斯）。。

Quantitative real-time PCR (qRT-PCR)

实时定量PCR（qRT-PCR）

Total RNA was extracted from patient tissue or cell lines using TRIzol, according to the manufacturer’s instructions, and reverse transcription was performed using an RNA reverse transcription kit. Real-time qRT-PCR analysis was performed using PowerUp SYBR Green premix (Thermo, USA), according to the manufacturer’s instructions, with three replicate wells for each sample, reaction data were exported, and the mRNA expression of the target gene calculated according to the detected Ct value as 2.

根据制造商的说明，使用TRIzol从患者组织或细胞系中提取总RNA，并使用RNA逆转录试剂盒进行逆转录。根据制造商的说明，使用Power Up SYBR Green premix（Thermo，USA）进行实时qRT-PCR分析，每个样品有三个重复孔，输出反应数据，并根据检测到的Ct值计算靶基因的mRNA表达为2。

−ΔΔCt

−DDCt

. Primer sequences used in this study are shown in Supplementary Table

本研究中使用的引物序列见补充表

S1级

Western blot (WB)

蛋白质印迹（WB）

Tissues or cell pellets were homogenized in RIPA buffer. Protein concentration was determined using a BCA protein assay kit (ThermoScience, Shanghai, China). Equal amounts of protein were separated by SDS-PAGE and then transferred to polyvinylidene difluoride membranes (Bio-Rad, Shanghai, China). Membranes were blocked with 5% skim milk for 1 h at room temperature, then primary antibodies were added and incubated overnight at 4 °C in a refrigerator.

将组织或细胞沉淀在RIPA缓冲液中匀浆。使用BCA蛋白质测定试剂盒（ThermoScience，Shanghai，China）测定蛋白质浓度。通过SDS-PAGE分离等量的蛋白质，然后转移到聚偏二氟乙烯膜（Bio-Rad，中国上海）上。在室温下用5%脱脂奶将膜封闭1小时，然后加入一抗并在冰箱中于4℃温育过夜。

The membranes were washed three times with TBST buffer solution for 10 min each, secondary antibodies added and incubated at room temperature for 1 h, then discarded, and washed three times with TBST buffer solution for 10 min each. After adding ultra-sensitive ECL chemiluminescent working solution (avoiding bubbles), blots were imaged using a chemiluminescent system, photographed, and analyzed.The full gel and blot images for this study are provided in an additional file (please refer to Additional File 1)..

将膜用TBST缓冲溶液洗涤三次，每次10分钟，加入二抗并在室温下孵育1小时，然后丢弃，并用TBST缓冲溶液洗涤三次，每次10分钟。加入超灵敏ECL化学发光工作溶液（避免气泡）后，使用化学发光系统对印迹进行成像，拍照和分析。本研究的完整凝胶和印迹图像在附加文件中提供（请参阅附加文件1）。。

Mass spectrometry

质谱法

Successfully transfected HCC cells were immunoprecipitated using anti-DYKDDDDK magnetic agarose (ThermoScience, Shanghai, China), washed five times with PBS for 10 min each, then resuspended in 2× SDS loading buffer, and boiled for 10 min in a water bath before gel electrophoresis. Mass spectrometry analysis was performed after staining with Coomassie brilliant blue..

使用抗DYKDDDDK磁性琼脂糖（ThermoScience，Shanghai，China）免疫沉淀成功转染的HCC细胞，用PBS洗涤五次，每次10分钟，然后重悬于2x SDS上样缓冲液中，并在水浴中煮沸10分钟。凝胶电泳前。用考马斯亮蓝染色后进行质谱分析。。

Ubiquitination assay

泛素化测定

Successfully transfected HCC cells were treated with MG132 for 4 h, then NP-40 lysis buffer was added to lyse the cells. Samples were incubated overnight at 4°C with an HA tag antibody, then Protein A/G PLUS-Agarose was added. After 6 h, the immune complex was collected and centrifuged at 804 RCF for 5 min at 4 °C.

成功转染的HCC细胞用MG132处理4小时，然后加入NP-40裂解缓冲液裂解细胞。将样品与HA标签抗体在4°C孵育过夜，然后加入蛋白A/G加琼脂糖。6小时后，收集免疫复合物并在4℃下以804 RCF离心5分钟。

The precipitates were collected and analyzed by WB..

收集沉淀物并通过WB分析。。

Protein half-life assay

蛋白质半衰期测定

HCC cells were treated with 20 μg/ml cycloheximide to block protein synthesis. Proteins were extracted after 0, 4, 8, 12, and 16 h, and the expression of related proteins was analyzed by WB.

用20μg/ml环己酰亚胺处理HCC细胞以阻断蛋白质合成。在0,4,8,12和16小时后提取蛋白质，并通过WB分析相关蛋白质的表达。

MeRIP-qPCR

HCC cells were treated with TRIzol to extract intracellular RNA. Anti-m6A or IgG was bound to protein A/G magnetic beads. Then, 100 μg of previously extracted RNA was mixed with the prepared protein A/G magnetic beads and eluted twice with N6-methyladenosine 5’-monophosphate sodium salt at 4 °C for 1 h each time, followed by qRT-PCR..

用TRIzol处理HCC细胞以提取细胞内RNA。抗m6A或IgG与蛋白A/G磁珠结合。然后，将100μg先前提取的RNA与制备的蛋白质A/g磁珠混合，并用N6-甲基腺苷5'-单磷酸钠盐在4℃下洗脱两次，每次1小时，然后进行qRT-PCR。。

Actinomycin D assay

放线菌素D测定

Each group of cells was treated with actinomycin D to inhibit transcription. Subsequently, RNA was extracted from each group and tested by qRT-PCR.

每组细胞用放线菌素D处理以抑制转录。随后，从每组中提取RNA并通过qRT-PCR测试。

Tumor formation in nude mice

裸鼠的肿瘤形成

Huh7 HCC cells with USP15 knocked down (Huh7-shUSP15) and wild-type Huh7 cells were used for these experiments. BALB/c-nu nude mice (

将USP15敲低的Huh7 HCC细胞（Huh7-shUS15）和野生型Huh7细胞用于这些实验。BALB/c-nu裸鼠(

= 20, 5 weeks old, male) were purchased from Weitonglihua Laboratory Animal Technology Co., Ltd. (Beijing, China) and randomly divided into four groups (

=20，5周大，雄性）购自北京威通利华实验动物技术有限公司，随机分为四组(

= 5 mice per group), to investigate whether USP15 promotes HCC progression and whether USP15 can enhance HCC resistance to lenvatinib. No blinding methods were employed in this experiment. Both cell lines were suspended in PBS and inoculated subcutaneously into nude mice using a fine needle. Once tumor volume reached 0.5 cm.

每组5只小鼠），以研究USP15是否促进HCC进展以及USP15是否可以增强HCC对lenvatinib的耐药性。本实验未采用致盲方法。将两种细胞系悬浮于PBS中，并使用细针皮下接种到裸鼠中。一旦肿瘤体积达到0.5厘米。

, the treatment groups received daily injections of lenvatinib (50 mg/kg) via the tail vein. After 25 days, mice were euthanized and the tumors were excised. Tumor size was measured with a vernier caliper, and tumor volume was calculated. For further immunohistochemistry, tumors were embedded in paraffin.

，治疗组每天通过尾静脉注射lenvatinib（50 mg/kg）。25天后，对小鼠实施安乐死并切除肿瘤。用游标卡尺测量肿瘤大小，并计算肿瘤体积。为了进一步的免疫组织化学，将肿瘤包埋在石蜡中。

All animal experiments were performed in accordance with the official guidelines of the Animal Ethics Committee..

所有动物实验均按照动物伦理委员会的官方指南进行。。

Other assays

其他测定

Details on other experimental methods, including immunohistochemistry, transfection, co-immunoprecipitation, cell growth assay, colony formation assay, and spheroid formation assay are provided in Additional File 2.

附加文件2中提供了其他实验方法的详细信息，包括免疫组织化学，转染，免疫共沉淀，细胞生长测定，集落形成测定和球体形成测定。

Statistical analysis

统计分析

All statistical data are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism v8.0 (GraphPad, Inc., USA) and Statistical Package for Social Sciences v22.0 (SPSS, Inc., Chicago, IL, USA).

所有统计数据均表示为平均±标准差。使用GraphPad Prism v8.0（GraphPad，Inc.，USA）和社会科学统计软件包v22.0（SPSS，Inc.，Chicago，IL，USA）进行统计分析。

< 0.05 was considered statistically significant.

<0.05被认为具有统计学意义。

Data availability

数据可用性

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

本研究中使用和/或分析的数据集可根据合理要求从通讯作者处获得。

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Acknowledgements

致谢

Not applicable.

不适用。

Funding

资金

This work was funded by the project of Health Science and Technology Capacity Improvement of Jilin Province (2023JC013). This work is supported by the Open Project Program of Key Laboratory of Early Prevention and Treatment for Regional High Frequency Tumor, Ministry of Education, Guangxi Medical University (Grant No.

这项工作由吉林省卫生科技能力提升项目（2023JC013）资助。这项工作得到了广西医科大学教育部区域高频肿瘤早期预防与治疗重点实验室开放项目的支持（批准号：。

GKE-KF202203)..

GKE-KF202203）。。

Author information

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Authors and Affiliations

作者和隶属关系

Department of Hepatobiliary Pancreatic Surgery, The Second Hospital of Jilin University, Changchun, China

吉林大学第二医院肝胆胰外科，长春

Ronghuan Fang, Zhigang Jia, Yuhang Xin, Kai Zhao, Yongsheng Yang & He Fang

方荣焕、贾志刚、余杭欣、赵凯、杨永胜和何芳

Jilin Engineering Laboratory for Translational Medicine of Hepatobiliary and Pancreatic Diseases, Changchun, China

吉林省肝胆胰疾病转化医学工程实验室，长春

Ronghuan Fang & Yuhang Xin

方荣焕和余杭欣

Department of Hepatobiliary Surgery, Afliated Hospital of Jining Medical, Jining, China

济宁医学院附属医院肝胆外科

Wei Qin & Haoran Lu

魏琴、陆浩然

Guangxi Key Laboratory of Early Prevention and Treatment for Regional High Frequency Tumor, Nanning, China

Yi Zhou

易州（Yi Zhou）

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Ronghuan Fang

方荣焕

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Zhigang Jia

贾志刚

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Yuhang Xin

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Kai Zhao

赵凯

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Wei Qin

魏琴（音）

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Haoran Lu

陆浩然

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Yi Zhou

易州（Yi Zhou）

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Yongsheng Yang

杨永生

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He Fang

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Contributions

捐款

YY, HF, and RF developed the original hypothesis, designed the experiments, and wrote the manuscript. RF, YX and ZJ performed in vivo and in vitro experiments. RF, KZ, WQ, HL, and YZ analyzed the data. YY supervised the study. All authors reviewed and approved the manuscript.

YY，HF和RF开发了原始假设，设计了实验并撰写了手稿。RF，YX和ZJ进行了体内和体外实验。RF，KZ，WQ，HL和YZ分析了数据。YY监督了这项研究。所有作者都审查并批准了手稿。

Corresponding authors

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通信对象

Yongsheng Yang

杨永生

或

He Fang

何芳（音）

Ethics declarations

道德宣言

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

All human samples involved in this study were reviewed and approved by the Ethics Committee of the Second Hospital of Jilin University. Animal studies were reviewed and approved by the Animal Ethics Committee of Changchun Weishi Testing Technology Service Co., Ltd.

吉林大学第二医院伦理委员会审查并批准了本研究涉及的所有人类样本。动物研究经长春伟世检测技术服务有限公司动物伦理委员会审查批准。

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Not applicable.

不适用。

Additional information

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Publisher’s note

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Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Springer Nature在已发布的地图和机构隶属关系中的管辖权主张方面保持中立。

Supplementary information

补充信息

41420_2024_2282_MOESM1_ESM.png

qRT-PCR and WB detection of differences in USP15 expression between the immortalized human liver cell line and the HCC cell lines

qRT-PCR和WB检测永生化人肝细胞系和HCC细胞系USP15表达的差异

41420_2024_2282_MOESM2_ESM.png

Lentivirus transfection to construct a Hep-3B cell line stably overexpressing USP15 and Huh-7 and HCC-LM3 cell lines with USP15 knocked down

慢病毒转染构建稳定过表达USP15和Huh-7的Hep-3B细胞系以及USP15敲除的HCC-LM3细胞系

Schematic diagram illustrating the role and mechanism of USP15

USP15的作用和机制示意图

Primer sequences in the study

研究中的引物序列

Relationship between USP15 and clinicopathological parameters in 52 HCC patients

52例HCC患者USP15与临床病理参数的关系

The full gel and blot images in this study

本研究中的完整凝胶和印迹图像

Details on other experimental methods

其他实验方法的详细信息

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引用本文

Fang, R., Jia, Z., Xin, Y.

方，R.，贾，Z.，辛，Y。

et al.

等人。

N6-methyladenosine-modification of USP15 regulates chemotherapy resistance by inhibiting LGALS3 ubiquitin-mediated degradation via AKT/mTOR signaling activation pathway in hepatocellular carcinoma.

USP15的N6-甲基腺苷修饰通过在肝细胞癌中通过AKT/mTOR信号传导激活途径抑制LGALS3泛素介导的降解来调节化疗耐药性。

Cell Death Discov.

细胞死亡发现。

, 3 (2025). https://doi.org/10.1038/s41420-024-02282-y

3 (2025).https://doi.org/10.1038/s41420-024-02282-y

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Received

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26 August 2024

2024年8月26日

Revised