miR-181d-5p对人结肠癌细胞凋亡的影响及机制

Effect and Mechanism of MicroRNA-181d-5p on Apoptosis of Human Colon Cancer Cells

目的:探讨微小RNA-181 d-5 p(microRNA miR-181 d-5 p)对人结肠癌细胞凋亡的影响及机制。方法:采用miR-181d-5p抑制物转染人结肠癌细胞系HCT116细胞,流式细胞术检测细胞凋亡情况,Real-time PCR和Western blot技术检测细胞miR-181 d-5 p、第10染色体同源缺失性磷酸酶-张力蛋白基因(PTEN)、黏附斑激酶(FAK)、B细胞淋巴瘤-2(Bcl-2)、半胱天冬蛋白酶3(Caspase-3)mRNA和PTEN、FAK、p-FAK、Bcl-2、激活型Caspase-3(cleaved Caspase-3)蛋白表达,Targetscan软件在线预测及双荧光素酶实验分析miR-181 d-5 p和PTEN的调控关系。结果:与阴性对照组比较,miR-181d-5p抑制物组miR-181 d-5 p mRNA表达明显下调(P<0.01),凋亡细胞明显增加(P<0.01),PTENmRNA表达水平明显上调(P<0.01),Bcl-2 mRNA表达水平明显下调(P<0.01),PTEN、cleaved Caspase-3蛋白表达水平明显升高(P<0.01),p-FAK、Bcl-2蛋白表达水平显著下调(P<0.01),其余各组之间差异均无统计学意义(P>0.05);Targetscan软件预测显示PTEN基因3′UTR含有miR-181 d-5 p的结合位点,双荧光素酶结果显示与突变型PTEN 3′UTR联合miR-181 d-5p模拟物组相比,野生型PTEN 3′UTR联合miR-181d-5p模拟物组荧光素酶活性明显降低,差异有高度统计学意义(P<0.01)。结论:miR-181 d-5 p可通过靶向调控PTEN抑制人结肠癌细胞凋亡,可成为结肠癌临床分子靶向治疗的潜在靶点。

Objective:To investigate the effect and mechanism of miR-181d-5p on apoptosis of human colon cancer cells.Methods:Human colon cancer cell line HCT116 was transfected with miR-181d-5p inhibitor.Apoptosis was detected by flow cytometry.Real-time PCR and Western blot were adopted to detect the protein expression of miR-181d-5p,PTEN,FAK,Bcl-2,Caspase-3 mRNA and PTEN,FAK,p-FAK,Bcl-2,cleaved Caspase-3 proteins.Targetscan analysis software online prediction and dual luciferase reporter gene system was used to analyze the regulatory relationship of miR-181d-5p and PTEN.Results:Compared with the negative control group,the expression of miR-181d-5p mRNA was down-regulated in the miR-181d-5p inhibitor group(P<0.01),the apoptotic cells were significantly increased(P<0.01),and the PTEN mRNA expression level was significantly up-regulated(P<0.01);Bcl-2 mRNA expression level was significantly down-regulated(P<0.01),PTEN and cleaved Caspase-3 protein expression levels were significantly increased(P<0.01),p-FAK,Bcl-2 protein expression levels were significantly down-regulated(P<0.01);the differences between the groups were not statistically significant(P>0.05).Targetscan software predicted that the 3'UTR of PTEN gene contained the binding site of miR-181d-5p,and the dual luciferase results showed that compared with the mutant PTEN 3'UTR+miR-181d-5p mimic group,the luciferase activity of the wild PTEN 3'UTR+miR-181d-5p mimic group was significantly lower,difference was statistically significant(P<0.01).Conclusion:miR-181d-5p may inhibit the apoptosis of human colon cancer cells by targeting PTEN,which could be the potential target of clinical molecular targeted therapy for treating colon cancer.