IGF2BP2/m6A/ITGA5信号轴调控肾透明细胞增殖和迁移

doi:10.6040/j.issn.1671-7554.0.2022.0429

摘要/Abstract

摘要： 目的探讨N6-甲基腺嘌呤(m6A)“识别蛋白”胰岛素样生长因子2 mRNA结合蛋白2(IGF2BP2)在肾透明细胞癌(ccRCC)中的潜在作用。方法通过分析癌症基因数据库(TCGA)、采用qRT-PCR法和Western blotting法检测IGF2BP2在肾癌中的表达水平,甲基化RNA免疫沉淀qPCR结合生物信息学鉴定整合素α5(ITGA5)mRNA的m6A修饰。采用基因敲低/过表达技术,采用qRT-PCR和Western blotting法检测IGF2BP2对ITGA5表达调控的作用。通过功能性获得和缺失实验确定IGF2BP2和ITGA5对肾癌细胞增殖和迁移的调控。结果 TCGA肾癌数据库显示,IGF2BP2在肾癌组织中高表达。同时高表达的IGF2BP2不利于患者的整体生存期(HR=1.6,P=0.005)和无病生存期(HR=1.9,P=0.014)。qRT-PCR法以及Western blotting法检测结果显示,IGF2BP2在肾癌组织中高表达。Western blotting检测结果显示,正常肾上皮细胞中IGF2BP2的表达低于其他几种肾癌细胞系。基因集合富集分析(GSEA)发现ITGA5可能是IGF2BP2下游靶分子。进一步通过敲除或者过表达发现,IGF2BP2在转录后水平调控ITGA5。甲基化RNA免疫沉淀qPCR发现ITGA5在3'UTR区存在m6A修饰化位点,低m6A修饰可能有助于维持其mRNA的稳定性。结论 IGF2BP2在肾透明细胞癌中高表达;IGF2BP2不利于肾癌患者的生存预后;IGF2BP2/ m6A/ ITGA5信号轴调控肾癌细胞的增殖、迁移和血管新生。

关键词: 肾透明细胞癌, 胰岛素样生长因子2 mRNA结合蛋白2, N6甲基腺苷, 增殖, 迁移, 血管新生

Abstract: Objective To explore the potential role of m6A “recognition protein” insulin-like growth factor 2 mRNA binding protein(IGF2BP2)in clear cell renal cell carcinoma(ccRCC). Methods The expression of IGF2BP2 in ccRCC was searched in cancer gene database TCGA and analyzed with qRT-PCR and Western blotting. The m6A modification of ITGA5 mRNA was identified with methylated RNA immunoprecipitation qPCR combined with bioinformatics. IGF2BP2 on the regulation of ITGA5 expression was detected with gene knockdown/overexpression experiments, qRT-PCR and Western blotting. The biological roles of IGF2BP2 and ITGA5 in ccRCC cells were determined with gain- and loss-of-function experiments. Results Data from TCGA showed that IGF2BP2 was highly expressed in ccRCC, while the high expression was not conducive to the overall survival(HR=1.6, P=0.005)and disease-free survival(HR=1.9, P=0.014). Western blotting and qRT-PCR also indicated that IGF2BP2 was highly expressed. Western blotting revealed that IGF2BP2 expression was significantly higher in ccRCC cell lines than in normal renal epithelial cells. Gene set enrichment analysis(GSEA)showed that ITGA5 might be a downstream target of IGF2BP2. Further knockdown or overexpression suggested that IGF2BP2 regulated ITGA5 at the post-transcriptional level. Methylated RNA immunoprecipitation qPCR showed that m6A modification sites were in the 3'UTR region of ITGA5, and low m6A modifications helped to maintain the stability of mRNA. Conclusion IGF2BP2 is upregulated in ccRCC, which is unfavorable for the prognosis and survival. IGF2BP2/m6A/ITGA5 signal axis regulates the proliferation, migration and angiogenesis in ccRCC.

Key words: Clear cell renal cell carcinoma, Insulin-like growth factor 2 mRNA binding protein, N6-methyladenosine, Proliferation, Migration, Angiogenesis

中图分类号:

R737.1

张振伟,李佳,陈克明. IGF2BP2/m6A/ITGA5信号轴调控肾透明细胞增殖和迁移[J]. 山东大学学报 (医学版), 2022, 60(9): 74-84.

ZHANG Zhenwei, LI Jia, CHEN Keming. IGF2BP2/m6A/ITGA5 signal axis regulates the proliferation and migration of renal clear cells[J]. Journal of Shandong University (Health Sciences), 2022, 60(9): 74-84.

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