miR-21在肾透明细胞癌中的表达及对增殖和凋亡的影响

Expression of miR-21 in renal clear cell carcinoma and its effects on cell proliferation and apoptosis

目的探讨miR-21在肾透明细胞癌中的表达及其临床意义,以及如何通过调节程序性细胞死亡因子4(programmed cell death 4,PDCD4)的表达影响786-O肾透明细胞癌细胞系的增殖和凋亡。方法通过分析The Cancer Genome Atlas(TCGA)肾透明细胞癌数据库,比较癌组织及正常癌旁组织中miR-21的表达水平;分析miR-21表达水平在不同临床病理分期肾癌组织中的差异;采用Kaplan-Meier法和对数秩和检验(Log-rank test)研究miR-21表达水平和患者生存之间的关系;通过转染miR-21抑制性核苷酸(AS-miR-21)下调miR-21表达水平,采用MTT和流式细胞术分别检测细胞增殖和凋亡,采用实时定量PCR(qRT-PCR)和Western blot测量PDCD4mRNA和蛋白质表达水平变化,采用双荧光素报告系统检测miR-21对PDCD4的直接调节。结果肾透明细胞癌组织中miR-21的表达水平显著高于癌旁组织(P<0.000 1)。miR-21在Ⅲ期和Ⅳ期肾癌组织中表达水平显著高于Ⅰ期(P均<0.000 1),miR-21表达水平与临床病理分期呈正相关(r=0.262,P<0.000 1)。miR-21表达水平与T分期(r=0.250,P<0.000 1)与淋巴结转移阳性(N1)以及远处转移均呈正相关(P均<0.001)。生存分析显示miR-21高表达患者中位生存时间显著短于miR-21低表达者中位生存时间(Log-rank,P<0.001)。下调miR-21后,786-O细胞的增殖能力较对照显著降低(P<0.05),凋亡显著增加(P=0.005),PDCD4mRNA(P=0.002)和蛋白质表达水平显著增高。双荧光素报告实验显示在转染AS-miR-21的细胞内PDCD4相对荧光强度较对照细胞显著升高(P=0.003)。结论 miR-21在肾透明细胞癌组织中表达升高,与患者临床病理分期呈正相关,和患者生存呈负相关;miR-21可能通过调节PDCD4表达水平,参与调节肾透明细胞癌细胞的增殖和凋亡。

Objective To investigate the expression of miR-21 in renal clear cell carcinoma and its clinical significance as well as how miR-21 regulates the proliferation and apoptosis of 786-O renal clear cell carcinoma cell line through regulating programmed cell death 4（PDCD4）.Methods By analyzing the data of renal clear cells cancer in The Cancer Genome Atlas（TCGA）database,we compared the expression of miR-21 in renal cancer tissues and adjacent normal tissues and explored the differences in miR-21 level in renal cancer at different clinicopathological stage,T stage,N stage and M stage.We also analyzed the association between miR-21 level and survival of patients by Kaplan-Meier method and Log-rank test.786-O cells were transfected with AS-miR-21 to deplete miR-21. MTT assay and flow cytometry were applied to measure cell proliferation and apoptosis,respectively.We then measured the mRNA and protein levels of PDCD4in786-O cells depleted for miR-21 by qRTPCR and Western blot,respectively,and performed a dual-luciferase assay to detect the direct regulation of PDCD4 by miR-21.Results Expression of miR-21 was significantly higher in renal cancer tissues than in adjacent tissues（P〈0.000 1）.The expression levels of miR-21 at stageⅢ and stageⅣ renal cancer were significantly higher than that at stageⅠ（both P〈0.000 1）.Moreover,miR-21 expression was positively correlated with clinicopathological stages of renal cancer by correlation analysis（r=0.262,P〈0.000 1）.The correlation test indicated that miR-21 level was also positively correlated with T stage of renal cancer（r=0.250,P〈0.000 1）,lymph node metastasis（N1）and distant metastasis（all P〈0.000 2）.Patients with high miR-21 expression had significantly shorter median survival time than those with low miR-21 expression（Log-rank P〈0.001）.Compared with control cells,786-O cells depleted for miR-21 showed significantly decreased cell proliferation（P〈0.05）and increased cell apoptosis rate（P=0.005）.PDCD4 mRNA（P=0.002）and protein levels were significantly elevated in 786-O cells with down-regulated miR-21 levels.In addition,the dual-luciferase reporter assay showed that the relative luciferase intensity of PDCD4 reporter in cells transfected with AS-miR-21 was significantly higher than that of control cells（P=0.003）.Conclusion miR-21 expression was up-regulated in renal cancer and correlated with clinicopathological stage and survival of patients.miR-21 promoted 786-O cell proliferation and inhibited apoptosis probably through regulating PDCD4 expression.These results indicate that miR-21 plays an important role in formation and development of renal cancer.