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

摘要：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PDCD4 mRNA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<0.0001)。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<0.0001)，miR-21表达水平与临床病理分期呈正相关(r=0.262，P<0.0001)。miR-21表达水平与T分期(r=0.250，P<0.0001)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)，PDCD4 mRNA(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。
ABSTRACT: 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 PDCD4 in 786-O cells depleted for miR-21 by qRT-PCR 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<0.0001). The expression levels of miR-21 at stage Ⅲ and stage Ⅳ renal cancer were significantly higher than that at stage Ⅰ (both P<0.0001). Moreover, miR-21 expression was positively correlated with clinicopathological stages of renal cancer by correlation analysis (r=0.262, P<0.0001). The correlation test indicated that miR-21 level was also positively correlated with T stage of renal cancer (r=0.250, P<0.0001), lymph node metastasis