microRNA-616在肝细胞癌侵袭转移中的作用机制研究

刘欣 许秋然 张美齐 蔡文伟 刘青光 涂建锋

microRNA-616在肝细胞癌侵袭转移中的作用机制研究

刘欣 许秋然 张美齐 蔡文伟 刘青光 涂建锋

目的 探讨microRNA-616（miR-616）在肝细胞癌（HCC）中的表达及其在HCC侵袭转移中的作用机制。方法通过qRT-PCR检测miR-616在60例HCC及癌旁组织中的表达，同时检测miR-616在不同肝癌细胞系中的表达情况；免疫组织化学染色检测miR-616在HCC及癌旁组织中波形蛋白（vimentin）、上皮型钙黏素（E-cadherin）及同源性磷酸酶-张力蛋白（PTEN）的表达情况；qRT-PCR检测miR-616在人正常永生化肝细胞（LO2）及5种不同肝癌细胞系（HepG2、SMMC-7721、Hep3B、Hu7）中的表达水平；使用miR-616抑制物作用Hep3B细胞，TranswellTM实验检测miR-616抑制后Hep3B细胞侵袭能力变化；应用miR-616抑制物及PTEN siRNA转染Hep3B细胞，qRT-PCR检测Hep3B中miR-616、vimentin、PTEN、E-cadherin的mRNA水平，Western blot法检测癌细胞中vimentin、PTEN、E-cadherin的蛋白水平。结果 miR-616在HCC组织中表达水平高于对应癌旁组织；miR-616在不同肝癌细胞系中表达均高于LO2细胞；miR-616高表达与低表达在血管侵犯、Edmonson-Steiner分级、TNM分期比较差异均有统计学意义(均P＜0.05)；miR-616高表达肝癌组PTEN及E-cadherin水平低于miR-616低表达肝癌组，而vimentin水平高于miR-616低表达肝癌组，相关性分析结果显示HCC组织中miR-616与E-cadherin、PTEN水平呈负相关，与vimentin水平呈正相关；在肝癌细胞中抑制miR-616后PTEN及E-cadherin表达水平上调，而vimentin表达降低，Hep3B细胞的侵袭能力明显降低。结论 miR-616在HCC组织中表达上调，其表达与HCC恶性临床病理特征有关，miR-616促进肝癌细胞侵袭转移的作用可能与其抑制PTEN表达及诱导上皮间质转化有关。

miR-616 肝细胞癌 侵袭 靶基因同源性磷酸酶-张力蛋白 上皮间质转化

【 Abstract】 Objective To investigate the expression of miR-616 in human hepatocellular carcinoma and its effects on tumor invasion and metastasis. Methods The expression of miR-616 was detected with real-time quantitative PCR(qRT-PCR) in 60 specimens of hepatocellular carcinoma (HCC)tissues and corresponding adjacent normal tissues;and also in human HCC HepG2,SMMC-7721,Hep3B,Hu75 cells and normal liver LO2 cells.The expression of vimentin,phosphatase,tensin homolog (PTEN)and E-cadherin was detected with immunohistochemical S-P method.The miR-616 inhibitor was transfected into Hep3B cells in vitro,cell invasion was analyzed by Transwell?assay.The expression of PTEN,E-cardhern and vimentin mRNAs and proteins after transfection was detected by qRT-PCR and Western blotting,respectively. Results The expression of miR-616 in HCC tissues was higher than that in matched tumor-adjacent normal tissues;and it was significantly correlated with venous infiltration,high Edmondson-Steiner grades and lymph node metastasis and TNM tumor stages in HCC.Elevated miR-616 expression was observed in HCC cell lines HepG2,SMMC-7721,Huh7 and Hep3B as compared with that in LO2 hepatic cell line.Furthermore,miR-616 expression was negatively correlated with E-cadherin and PTEN,positively correlated with vimentin in HCC tissues.The miR-616 inhibitor suppressed the migration and invasion in Hep3B cells;and it increased the expression of E-cadherin and PTEN,while decreased the expression of vimentin in Hep3B cells.In addition,down-regulation of PTEN' expression partially abrogated the effect of miR-616 inhibitor in HCC cells. Conclusion The expression of miR-616 in HCCtissues is significantly up-regulated,and it can promote proliferation and induce apoptosis of HCC cells by down-regulating the expressions of PTEN.

【 Key words】 microRNA-616 Hepatocellular carcinoma Migration and invasion Phosphatase and tensin homolog (PTEN) Epithelial-mesenchymal transition

肝细胞癌（hepatocellular carcinoma，HCC）是常见的恶性肿瘤之一，其已成为亟待解决的医学界问题[1]。目前在世界范围内HCC的治疗取得了一定的进展[2]，但是HCC发生、发展的具体分子机制仍不十分清楚。……