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目的探讨miR-134下调叉头盒蛋白M1(forkhead box M1,FOXM1)的机制及其影响肝细胞癌(hepatocellular carcinoma,HCC)细胞增殖的作用。方法应用CCK-8法检测miR-134对HCC细胞增殖的作用;经Western blot和Real-time PCR检测人正常肝细胞L02和5种HCC细胞系中FOXM1和miR-134的表达;用miR-134模拟物(miR-134 mimic)或miR-134抑制剂(miR-134 inhibitor)转染HCC细胞后,经Western blot和Real-time PCR检测FOXM1及其下游靶基因Cyclin D1的表达;应用生物信息学分析miR-134在人FOXM1 3’-UTR的可能结合位点,通过报告基因检测实验分析miR-134与FOXM1的3’-UTR的特异性结合;将miR-134 mimic和FOXM1表达质粒(无3’-UTR)共转染于HCC细胞后,经CCK-8法检测细胞的增殖情况。结果 miR-134可显著抑制HCC细胞增殖(P<0.05);与人正常肝细胞L02相比,miR-134在HCC细胞中的表达明显降低(P<0.05),而FOXM1蛋白表达明显增强,二者存在负相关(R2=0.705,P<0.05);miR-134可显著下调FOXM1蛋白及其下游靶基因Cyclin D1的表达(P<0.05);报告基因检测实验证实miR-134可特异性结合于FOXM1 mRNA的3’-UTR(P<0.01);细胞增殖实验检测结果显示,过表达缺失3’-UTR的FOXM1可显著减弱miR-134对HCC细胞增殖的抑制效应(P<0.05)。结论 miR-134通过靶向结合于FOXM1的3’-UTR而下调FOXM1蛋白的表达,从而抑制HCC细胞的增殖。
Objective To investigate the mechanism of miR-134 down-regulation of forkhead box M1 (FOXM1) and its effect on the proliferation of hepatocellular carcinoma (HCC) cells. Methods The effect of miR-134 on the proliferation of HCC cells was detected by CCK-8 method. The expression of FOXM1 and miR-134 in human normal hepatocytes L02 and 5 HCC cell lines was detected by Western blot and Real-time PCR. After transfected into HCC cells with miR-134 mimic or miR-134 inhibitor, the expression of FOXM1 and its downstream target gene Cyclin D1 was detected by Western blot and Real-time PCR. The bioinformatics The possible binding sites of miR-134 on the 3 ’-UTR of human FOXM1 were analyzed, and the specific binding of miR-134 to the 3’-UTR of FOXM1 was analyzed by reporter assay. The miR-134 mimic and FOXM1 expression plasmids ’-UTR) were co-transfected in HCC cells, the proliferation of cells by CCK-8 assay. Results miR-134 significantly inhibited the proliferation of HCC cells (P <0.05). Compared with normal human L02 cells, the expression of miR-134 in HCC cells was significantly decreased (P <0.05), while the expression of FOXM1 protein was significantly increased. (R2 = 0.705, P <0.05); miR-134 significantly down-regulated FOXM1 protein and its downstream target gene Cyclin D1 expression (P <0.05); reporter gene test confirmed that miR-134 can specifically bind to FOXM1 mRNA 3’-UTR (P <0.01). The results of cell proliferation assay showed that overexpression of FOXM1 lacking 3’-UTR significantly attenuated the inhibitory effect of miR-134 on HCC cell proliferation (P <0.05). Conclusions miR-134 down-regulates FOXM1 protein expression by binding to the 3’-UTR of FOXM1, thereby inhibiting the proliferation of HCC cells.