微小RNA-132对体内外肝癌细胞侵袭和转移的影响

In vitro and in vivo studies on the effects of microRNA-132 on invasion and metastasis in human liver cancer cells

目的观察微小RNA-132（miR-132）对体内外人肝癌细胞侵袭和转移的影响，初步探讨其作用机制。方法实时荧光定量逆转录一聚合酶链反应（实时-qPCR）检测miR-132在4种肝癌细胞株（MHCC97H、HCCLYH、MHCC97L和SMMC-7721）、人正常肝细胞株HL-7702以及20例发生转移的肝癌组织和25例未发生转移肝癌组织中的表达。采用划痕实验、Transwell小室实验和经裸鼠尾静脉注射肝癌细胞形成的肝癌肺转移模型检验转染miR-132后对体内外肝癌MHCC97H细胞侵袭和转移的影响。蛋白印迹检测体外MHCC97H细胞中钙黏蛋白E（E-cadherin）、钙黏蛋白α（α-eadhefin）、波形蛋白（vimentin）、纤连蛋白（fibronectin）和锌指E盒结合同源盒蛋白2（zincfingerE-boxbindingho-meoboxprotein2，ZEB2）蛋白的表达。免疫组织化学法检测肝癌肺转移灶组织中ZEB2的表达。结果miR．132在4种肝癌细胞株中的表达均明显低于人正常肝细胞（P〈0．05），在伴转移肝癌组织中miR-132的表达明显低于未转移肝癌组织，差异有统计学意义（P〈0．05）。在体外实验，转染组细胞中miR-132的表达明显升高，细胞迁移和侵袭明显抑制，与对照组相比较，差异有统计学意义（P〈0．05）。转染组细胞中E-cadherin和α-eadhefin蛋白表达上调，而vimentin、fibronectin和ZEB2蛋白的表达下调，与对照组相比较，差异均有统计学意义（P〈0．05）。在体内实验中，转染组裸鼠肺转移灶数目明显减少，ZEB2蛋白表达下调。结论miR-132对体内外肝癌细胞侵袭和转移有明显抑制作用，有望成为肝癌治疗的新靶点。

Objective To observe the biological role and the underlying mechanisms of miR-132 in liver cancer on invasion and metastasis. Methods Real-time quantitative polymerase chain reaction （ RT- qPCR） analysis was used to examine the expression of miR-132 in four liver cancer cell lines （ MHCC97H, HCCLYH, MHCC97L and SMMC-7721 ）, a normal liver cell line HL-7702, and in liver tumor tissues with or without metastases. The biological effects of miR-132 transfection on human liver can-cer cells were as- sessed by wound assay, matrigel counting and in vivo experiments in nude mice. Western blotting was used to detect the expression of E-cadherin, cL-cadherin, vimentin, fibronectin and ZEB2 in li-ver cancer cells. Immunohistochemistry was used to detect positive expression of ZEB2 in xenograft tumors. Results The expressions of miR-132 were downregulated in the four liver cancer cell lines when compared with the normal liver cell line （ P 〈 0.05 ）, and in the liver cancer tissues with distant metastases when compared with the tissues without metastases （ P 〈 0. 05）. After transfection, ectopic expressions of miR-132 markedly inhibited cell migration and invasion in liver cancer ceils. When compared with the control group, the expressions of E-cadherin and α-cadherin in the miR-132 transfection group were significantly increased, but the expressions of vimentin, fibronectin and ZEB2 were decreased. In addition, the numbers of metastatic lung lesions in nude mice in the miR-132 transfection group was markedly decreased when compared with the control group. The expressions of ZEB2 in the miR-132 transfection group was also significantly decreased when compared with the control group. Conclusions Transfection of miR-132 effectively inhibited invasion and metastasis of liver cancer cells in vitro and in vivo. miR-132 may become a new target for regulation of gene expression in liver cancer.