Study of molecular mechanism of miR-34c in the inhibition of endometrial carcinoma
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摘要: 目的 MicroRNAs (miRNAs)是内源性表达长度约为22个核苷酸的小RNA分子,通过结合到靶mRNA上而抑制基因的表达。近年的研究表明某些miRNAs可以调节肿瘤细胞的增殖与迁移,然而miRNA在子宫内膜癌中的作用有待于进一步探索。本研究旨在探讨miR-34c在人子宫内膜癌中的表达规律、功能和分子机制。 方法 通过荧光定量PCR技术检测20例人子宫内膜癌组织标本中miR-34c的表达。通过阳离子脂质体介导的方法将miR-34c转染入人子宫内膜癌细胞RL95-2中,应用MTS法检测子宫内膜癌细胞的增殖,克隆形成实验检测子宫内膜癌细胞的生长,流式细胞术检测细胞周期,Transwell实验检测子宫内膜癌细胞的迁移。生物信息学和荧光素酶报告基因实验确认miR-34c作用的靶基因。RNAi技术下调c-Met的表达。通过蛋白印迹检测miR-34c对c-Met蛋白以及细胞内重要信号通路与细胞周期相关蛋白的影响。 结果 miR-34c在子宫内膜癌中表达明显降低。miR-34c能使子宫内膜癌细胞RL95-2细胞周期滞留在G1期,显著抑制细胞的增殖。细胞移形实验发现miR-34c能够显著抑制RL95-2细胞迁移。生物信息分析表明c-Met是miR-34c作用的靶基因,进一步通过荧光素酶报告基因实验得以证实。蛋白印迹分析发现miR-34c能下调RL95-2细胞中的c-Met蛋白、p-Akt蛋白以及p-ERK1/2蛋白表达情况,同时还能够下调CDK4、CDK6蛋白以及p-Rb蛋白的表达水平。RNAi技术下调c-Met的表达能够明显抑制子宫内膜癌细胞的增殖与迁移。 结论 本研究结果证明miR-34c通过作用于靶基因c-Met而抑制子宫内膜癌细胞的增殖与迁移,说明miR-34c是子宫内膜癌中的重要抑癌基因。Abstract: Objective MicroRNAs(miRNAs) are endogenously expressed 22-nucleotide small RNAs which repress protein translation through binding to a target mRNA. The recent studies have shown that some miRNAs can regulate proliferation and migration of tumor cells. The role of miRNAs in endometrial carcinoma,however,warrants for further investigation. In the present study,we investigated miR-34 c expression pattern,function and molecular mechanism in endometrial carcinoma. Methods Real-time RT-PCR was performed to detect the expression of miR-34 c in 20 endometrial carcinoma specimens. The miR-34 c was transfected into RL95-2 cells by using lipofectamine reagent. The proliferation of RL95-2cells was examined by MTS cell proliferation assay and colony formation assay. The cell cycle was analyzed by flow cytometry and cell migration was evaluated by Transwell assay. The target of miR-34 c was predicted by bioinformatics and confirmed by luciferase assay. C-Met was down-regulated by RNAi. Western blot analysis was carried out to examine the effects of miR-34 c on c-Met and proteins in important cellular signaling pathways. Results MiR-34 c was dramatically down-regulated in most endometrial carcinoma samples. Ectopic miR-34 c can inhibit the proliferation of RL95-2 cells through cell cycle G1 arrest. Furthermore,miR-34 c suppressed RL95-2 cell migration. Bioinformatics revealed c-Met was a target gene of miR-34 c and confirmed by luciferase assay. Western blot analysis indicated that miR-34 c down-regulated the expression of c-Met,p-Akt,p-ERK1/2,CDK4,CDK6 and p-Rb. Conclusion The miR-34 c can inhibite endometrial carcinoma cell proliferation and migration by targeting c-Met,which indicates that miR-34 c may act as an important tumor suppressor in ndometrial carcinoma.
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Key words:
- MiR-34c /
- Endometrial carcinoma /
- Proliferation /
- Migration /
- C-Met
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