miR-222-3p通过靶向调节Bim蛋白表达参与肾细胞癌的发生发展

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摘要目的: 检测miR2223p在肾细胞癌组织中的表达，探讨其参与肾细胞癌发生发展的分子机制。方法: 运用qRTPCR检测15对肾细胞癌组织和对应癌旁组织miR2223p的表达水平。生物信息学预测miR2223p的靶基因，荧光素酶报告实验验证其靶向调控作用。运用免疫组化和蛋白质印迹技术检测癌组织中靶基因蛋白表达水平。进一步通过脂质体瞬时转染技术实现肾细胞癌细胞株769P中miR2223p过表达，采用流式细胞术检测miR2223p对细胞凋亡的影响。结果： qRTPCR结果显示，miR2223p在肾细胞癌组织的表达水平显著高于癌旁组织（P<0.01）。生物信息学预测显示，抗凋亡蛋白Bim是miR2223p潜在的靶基因。荧光素酶报告实验证实，过表达miR2223p可抑制含有Bim 3’UTR的荧光素酶报告基因的荧光素酶活性，但对Bim 3’UTR突变体的荧光素酶活性没有影响。免疫组化结果显示，肾细胞癌组织中Bim蛋白免疫组化阳性反应程度明显低于相应的癌旁组织（P<0.01）；蛋白质印迹结果进一步证实Bim蛋白在肾细胞癌组织中的表达较癌旁组织明显下调（P<0.01）。进一步在769P细胞中瞬时过表达miR2223p后，发现Bim蛋白水平明显下调（P<0.01），但Bim mRNA水平无明显变化。流式细胞术检测结果显示，过表达miR2223p可显著抑制769P细胞的凋亡（P<0.01）。结论： miR2223p可通过靶向作用于抑癌基因Bim表达抑制肾癌细胞的凋亡，在肾癌的发生发展中起重要作用，miR2223p/Bim轴为肾细胞癌治疗提供新的途径。

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关键词 ：肾细胞癌, miR-222-3p, Bim, 凋亡

Abstract：Objective: To detect the expression of miR2223p in renal cell carcinoma(RCC) tissues, and explore its role and mechanisms in the development and progression of RCC. Methods: Total RNA was extracted from the tissues of 15 pairs of RCC and their adjacent tissues. The expression of miR2223p was determined by realtime fluorescent quantitative PCR（qRTPCR）. The target genes of miR2223p were predicted by bioinformatics and verified by luciferase reporter gene assay. Immunohistochemistry and Western blotting analysis were used to detect the expression of target gene in RCC tissue samples. Lipofectamine 2000 was used to overexpress miR2223p in renal carcinoma cell line 769P. The apoptosis of 769P cells was assessed by using Annexin VFITC and propidium iodide staining. Results: qRTPCR results showed that miR2223p was significantly higher in RCC tissue samples than in adjacent tissues(P<0.01). Bim was predicted as a potential target gene of miR2223p by bioinformatics methods. The luciferase reporter gene assay results showed that overexpression of miR2223p inhibited the Bim 3′UTR activity, whereas did not affect the luciferase activity of mutant form of Bim 3′UTR. Immunohistochemistry assay showed that the staining intensity of Bim was lower in RCC tissues than in matched adjacent tissues(P<0.01).Western blotting analysis showed that Bim was significantly downregulated in RCC tissues(P<0.01).Transient overexpression of miR2223p in 769P cells resulted in reduction of Bim protein levels(P<0.01); however, Bim mRNA level did not change significantly. In vitro cell function studies showed that overexpression of miR2223p significantly inhibited the apoptosis of 769P cells(P<0.01). Conclusion: miR2223p could cause carcinogenesis via inhibiting the apoptosis of renal cell carcinoma, which plays an important role in the development and progression of RCC through targeting tumor suppressor gene Bim. The miR2223p/Bim axis may potentially serve as a novel therapeutic application for RCC.

收稿日期: 2017-08-31

基金资助:

国家自然科学基金资助项目(81472021，81772282)；江苏省自然科学基金资助项目（BK20140730）；生命分析化学国家重点实验室资助项目（5431ZZXM1601）

通讯作者: 张春妮（通讯作者），主任技师，博士生导师，E-mail：zchunni27@hotmail.com

作者简介: 田亚萍（1989—），女，硕士研究生；

引用本文:

田亚萍1，2，王成2，钟锦莎2，丁梦2，葛京平3，张春妮2. miR-222-3p通过靶向调节Bim蛋白表达参与肾细胞癌的发生发展[J]. 江苏大学学报:医学版, 2018, 28(01): 6-11. TIAN Ya-ping1,2， WANG Cheng2， ZHONG Jin-sha2， DING Meng2， GE Jing-ping3， ZHANG Chun-ni2. miR-222-3p is involved in the development and progression of renal cell carcinoma by targetedly inhibiting Bim protein expression. Journal of Jiangsu University(Medicine Edition), 2018, 28(01): 6-11.

链接本文:

http://zzs.ujs.edu.cn/xbyxb/CN/ 或 http://zzs.ujs.edu.cn/xbyxb/CN/Y2018/V28/I01/6

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