miR-134通过下调叉头盒蛋白M1抑制肝细胞癌细胞增殖

miR-134 suppresses proliferation in hepatocellular carcinoma cells by downregulating FOXM1

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 by which miR-134 regulates the expression of forkhead box M1 （FOXM1） and determine the effect of miR-134 on the proliferation of hepatocellular carcinoma cells （HCC）. Methods The proliferation of HCC cells was analyzed by CCK=8 assay. The levels of FOXM1 and miR-134 were separately examined by Western blotting and real-time PCR in human normal hepatic cell line L02 and 5 kinds of HCC cell lines. After transfection with miR-134 mimic or inhibitor, the expression of FOXM1 and its target gene CyclinD1 in HCC cells were measured by Western blotting and real-time PCR. The potential binding site of miR-134 on the 3′ UTR of human FOXM1 mRNA was predicted by using online bioinformative databases. Then the luciferase reporter assays were performed to identify the specific binding of miR-134 with 3′ UTR of FOXM1 mRNA. After cotransfected with miR-134 mimic or FOXM1-expressing plasmids （without 3′ UTR）, the HCC cell proliferation was detected by CCK-8 assay. Results miR-134 dramatically suppressed the proliferation of HCC cells （P〈0.05）. Compared to normal liver cell line, L02 cells, miR-134 was lowly expressed while the FOXM1 protein was highly expressed in the HCC cells（P〈0.05）, and a negative correlation was found between miR-134 level and FOXM1 protein level （R^2=0.705, P〈0.05）. miR-134 significantly downregulated FOXM1 protein, and its downstream target gene, CyclinD1, in HCC cells （P〈0.05）. The luciferase reporter assay showed that miR-134 specifically bound to the 3′UTR of FOXM1 mRNA （P〈0.01）. The cell proliferation assay revealed that the ectopic expression of FOXM1 without 3′UTR in HCC cells significantly attenuated the miR-134-mediated growth inhibition of HCC cells （P〈0.05）. Conclusion miR134 down-regulates FOXM1 protein by directly targeting the 3′ UTR of FOXM1 mRNA, and subsequently suppresses the proliferation of HCC cells.