甲基转移酶PRMT5稳定USP15促进乳腺癌发生发展的作用

doi:10.6040/j.issn.1671-7554.0.2023.0132

摘要/Abstract

摘要： 目的探讨蛋白精氨酸甲基转移酶5(PRMT5)通过甲基化作用,修饰并上调泛素特异性蛋白酶15(USP15)的蛋白水平,从而促进乳腺癌发生发展的作用。方法采用基因表达谱交互分析(GEPIA2)在线数据库和Kaplan-Meier Plotter生存曲线分析,分析PRMT5在肿瘤组织和正常组织中的表达差异,及其与乳腺癌患者临床预后的关系。采用免疫亲和纯化及银染联合质谱分析得到与PRMT5结合的蛋白。通过慢病毒稳转技术在MCF-7细胞中过表达或敲低PRMT5,实时荧光定量PCR(qRT-PCR)及蛋白免疫印迹(Western blotting)技术检测细胞内相关蛋白的表达。质粒或小干扰RNA(siRNA)转染技术敲低或过表达USP15, qRT-PCR及Western blotting技术检测细胞内相关蛋白表达,采用细胞活性实验(CCK8)、克隆形成和Transwell实验检测其对乳腺癌细胞增殖与侵袭能力的影响。利用EPZ015666(GSK3235025)小分子抑制剂抑制PRMT5甲基转移酶活性后,采用Western blotting技术检测细胞内相关蛋白的变化,采用CCK8和Transwell实验检测其对乳腺癌细胞增殖和侵袭能力的影响。结果生物信息学分析结果显示,PRMT5在乳腺癌患者中高表达并与乳腺癌患者不良预后相关。免疫亲和纯化及银染联合质谱分析并通过免疫共沉淀(Co-IP)实验验证,发现PRMT5与USP15存在相互结合。在乳腺癌细胞MCF-7中,过表达FLAG-PRMT5后,通过Western blotting和qRT-PCR实验检测,发现USP15的蛋白水平明显增加,而不影响USP15的mRNA水平;敲低PRMT5表达后,USP15的蛋白水平降低,而mRNA水平未发生改变。在MCF-7细胞中,过表达Myc-USP15明显降低FBXW7的mRNA及蛋白水平,细胞功能实验结果显示,细胞的增殖和侵袭能力增加,敲低USP15则结果相反。Western blotting和细胞功能实验结果显示,EPZ015666抑制PRMT5的甲基转移酶活性后,可通过降低USP15的蛋白水平降低细胞的增殖和侵袭能力。结论乳腺癌细胞中PRMT5与USP15相互结合,并以甲基转移酶活性依赖的方式上调USP15的蛋白水平。USP15的升高可通过催化组蛋白H2BK120ub的去泛素化修饰,使FBXW7基因转录激活被抑制,进而促进乳腺癌细胞增殖和上皮-间质转化过程等,促进乳腺癌的发生与进展,PRMT5是乳腺癌潜在的诊断及干预治疗靶点。

关键词: 乳腺癌, PRMT5蛋白, USP15蛋白, FBXW7蛋白, 增殖, 上皮-间质转化

Abstract: Objective To explore the mechanism by which protein arginine methyltransferase 5(PRMT5)up-regulates the expression of ubiquitin-specific protease 15(USP15)through methylation, thereby promoting the occurrence and development of breast cancer. Methods Gene Expression Profiling Interactive Analysis(GEPIA2)online database and Kaplan-Meier plotter survival curve analysis were used to explore the difference in the expression of PRMT5 in tumor tissue and normal tissue and its relationship with prognosis. The protein bound with PRMT5 was obtained with immunoaffinity purification and silver staining combined with mass spectrometry analysis. After PRMT5 was overexpressed or knocked down in MCF-7 cells with lentiviral stabilizing technology, the expressions of related proteins were detected with quantitative real-time PCR(qRT-PCR)and Western blotting. After USP15 was overexpressed or knocked down with plasmid or small interfering RNA(siRNA)transfection, the expressions of related proteins were determined with qRT-PCR and Western blotting, and the proliferation and invasion ability of breast cancer cells were detected with CCK8, colony formation and Transwell assay. After the activity of PRMT5 methyltransferase was inhibited with EPZ015666(GSK3235025)small molecule inhibitor, the changes of related proteins were determined with Western blotting, and the effects on the proliferation and invasion ability of breast cancer cells were assessed with CCK8 and Transwell assay. Results PRMT5 was highly expressed in breast cancer patients and was associated with poor prognosis. PRMT5 and USP15 interacted with each other. In breast cancer cell MCF-7, after overexpression of FLAG-PRMT5, the protein expression of USP15 was significantly increased, while the mRNA level remained unchanged. After the expression of PRMT5 was knocked down, the protein level of USP15 decreased, but the mRNA level did not change. In MCF-7 cells, overexpression of Myc-USP15 significantly decreased the mRNA and protein levels of FBXW7, but increased the proliferation and invasion ability, while knockdown of USP15 had the opposite results. After EPZ015666 inhibited the methyltransferase activity of PRMT5, the proliferation and invasion ability of cells were decreased by reducing the protein level of USP15. Conclusion PRMT5 interacts with USP15 in breast cancer cells and upregulates the protein level of USP15 in a methyltransferase activity-dependent manner. The increase of USP15 can inhibit the transcriptional activation of F-box and WD repeat domain-containing 7(FBXW7)gene by catalyzing the deubiquitination modification of histone H2BK120ub, promote breast cancer cell proliferation and epithelial to mesenchymal transition(EMT), and promote the occurrence and progress of breast cancer. These findings suggest that PRMT5 is a potential diagnostic and therapeutic target for breast cancer.

Key words: Breast cancer, PRMT5, USP15, FBXW7, Proliferation, Epithelial to mesenchymal transition

中图分类号:

R737.9

金珊,高杰,谢玉姣,展垚,杜甜甜,王传新. 甲基转移酶PRMT5稳定USP15促进乳腺癌发生发展的作用[J]. 山东大学学报 (医学版), 2023, 61(7): 1-11.

JIN Shan, GAO Jie, XIE Yujiao, ZHAN Yao, DU Tiantian, WANG Chuanxin. Effects of methyltransferase PRMT5 on stabilizing USP15 to promote the occurrence and development of breast cancer[J]. Journal of Shandong University (Health Sciences), 2023, 61(7): 1-11.

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