慢病毒转染调控FoxM1表达对人肝内胆管细胞癌增殖、侵袭及MMPs表达的影响

Effects of FoxM1 expression regulated by lentiviral transfection on the proliferation and invasion of human intrahepatic cholangiocarcinoma and the expression of MMPs

目的通过实验探讨慢病毒转染调控FoxM1表达对人肝内胆管细胞癌(ICC)增殖、侵袭能力及基质金属蛋白酶(MMP)-9和MMP-2表达的影响。方法Western blot检测ICC细胞株HCCC-9810、RBE及SSP-25的FoxM1蛋白表达水平,选取表达量较低者作为上调FoxM1细胞株,较高者作为下调细胞株;将分别携带FoxM1质粒和shRNA的慢病毒载体转染目标上调和下调ICC细胞株,建立稳定上下调FoxM1的细胞株(Western blot验证);MTT法检测转染后细胞增殖力,Transwell侵袭实验检测细胞侵袭能力;qPCR检测各组稳定转染细胞株MMP-9及MMP-2 mRNA表达水平。结果SSP-25的FoxM1蛋白表达最高,HCCC-9810最低,由此选取SSP-25作为下调FoxM1表达目标细胞株,HCCC-9810作为上调FoxM1表达目标细胞株。慢病毒转染成功构建稳定上调(HCCC-9810-FoxM1组)及下调FoxM1细胞株(SSP-25-shFoxM1组);HCCC-9810-FoxM1组增殖及侵袭能力明显高于HCCC-9810-Control组(均P<0.05),而SSP-25-shFoxM1组增殖及侵袭能力较SSP-25-Control组明显下降(均P<0.05);HCCC-9810-FoxM1组中MMP-9及MMP-2 mRNA表达较HCCC-9810-Control组明显升高,而SSP-25-shFoxM1组中MMP-9及MMP-2 mRNA表达较SSP-25-Control组明显降低(均P<0.01)。结论FoxM1促进ICC细胞增殖及侵袭,可能调控MMP-9及MMP-2表达,也有可能作为ICC潜在的生物标志物。

Objective To investigate the effects of FoxM1 regulated by lentiviral transfection on the proliferation and invasion of human intrahepatic cholangiocarcinoma(ICC)and the expression levels of MMP-9 and MMP-2 in vitro.Methods Western blot assay was used to detect the FoxM1 protein expression levels of ICC cell lines HCCC-9810,RBE and SSP-25.The higher expression level of cell line was selected as the down-regulated FoxM1 cell line,and the lower one was selected as the up-regulated FoxM1 cell line.FoxM1 plasmid and shRNA lentiviral vector transfection targeting up regulating and down-regulating ICC cell lines were established a cell line that stably up-regulating or down-regulating FoxM1 cells(verified by Western blot assay).MTT method was used to detect cell proliferation after transfection,and Transwell invasion test was used to detect cell invasion.qPCR was used to detect the expression levels of MMP-9 and MMP 2 mRNA in stably transfected cell lines of each group.Results The protein of FoxM1 expression of SSP-25 was the highest,and HCCC-9810 was the lowest.Therefore,SSP-25 was selected as the target cell line for down-regulating FoxM1 expression,and HCCC-9810 was selected as the target cell line for up-regulating FoxM1 expression.The lentiviral transfection successfully constructed stable overexpression(the HCCC-9810-FoxM1 group)and down-regulated FoxM1 cell line(the SSP-25-shFoxM1 group).The proliferation and invasion ability were significantly higher in the HCCC-9810-FoxM1 group than those of the HCCC-9810-Control group(P<0.05).The proliferation and invasion ability were significantly lower in the SSP-25-shFoxM1 group than those of the SSP-25-Control group(P<0.05).In the HCCC-9810-FoxM1 group,the mRNA expression levels of MMP-9 and MMP-2 were significantly higher than those of the HCCC-9810-Control group,while the mRNA expression levels of MMP-9 and MMP-2 were significantly lower in the SSP-25-shFoxM1 group than those of the SSP-25-Control group(P<0.05).Conclusion FoxM1 promotes the proliferation and invasion of ICC cells.FoxM1 may affect the expression levels of MMP-9 and MMP-2.FoxM1 may serve as a potential biomarker of ICC.