miR-130b在肝细胞癌中的表达及临床病理意义

The expression and clinopathological significance of miR-130b in human hepatocellular carcinoma

目的探讨miR-130b在人肝细胞癌(HCC)中的表达、与临床病理特征的关系及可能机制。方法用实时定量PCR(qRT-PCR)检测miR-130b在86例HCC及癌旁组织、不同肝癌细胞系的表达;免疫组织化学染色检测miR-130b不同表达水平的HCC组织中上皮钙黏素(E-cadherin)、波形蛋白(vimentin)及过氧化物酶体增殖物激活受体γ(PPARγ)的表达情况;qRT-PCR检测miR-130b在LO2人正常永生化肝细胞及Hep G2、Hep3B、SMMC-7721、Hu7肝癌细胞系的表达水平;应用人工合成的miR-130b抑制物转染SMMC-7721细胞,TranswellTM实验检测SMMC-7721细胞侵袭能力变化;应用人工合成的miR-130b抑制物及PPARγ小干扰RNA(siRNA)转染SMMC-7721人肝癌细胞,qRT-PCR检测癌细胞中miR-130b、PPARγ、E-cadherin、vimentin的mRNA水平,Western blot法检测癌细胞中PPARγ、E-cadherin、vimentin的蛋白水平。结果 miR-130b在HCC组织中表达水平显著高于对应癌旁组织;肝癌组织中miR-130b异常表达与门静脉侵犯、原发肿瘤分级、肿瘤TNM分期显著相关;miR-130b在不同肝癌细胞系中表达均高于LO2细胞;miR-130b高表达组PPARγ及E-cadherin蛋白水平显著低于miR-130低表达组,而vimentin水平显著高于miR-130b低表达组,相关性分析结果显示肝癌组织中miR-130b与PPARγ蛋白、E-cadherin蛋白水平呈显著负相关,与vimentin蛋白水平呈显著正相关;抑制miR-130b水平,可上调PPARγ蛋白的水平,E-cadherin蛋白的表达显著增加,而vimentin蛋白表达显著降低,SMMC-7721细胞的侵袭能力降低。PPARγsiRNA可部分逆转miR-130b抑制物对SMMC-7721细胞的作用。结论 miR-130b在HCC组织中表达上调并与HCC恶性临床病理特征有关,miR-130b可能通过抑制PPARγ表达及诱导上皮间质转化促进肝癌细胞侵袭。

Objective To investigate the expression of miR-130 b in human hepatocellular carcinoma（ HCC） and its correlations with clinical-pathological features. Methods Real-time quantitative PCR（ qRT-PCR） was applied to detect the expression of miR-130 b in HCC tissues（ n = 86）,matched normal tumor-adjacent tissues and 5 HCC cell lines（ LO2 human normal hepatocytes, Hep G2, Hep3 B, SMMC-7721, Hu7 cells）. The expressions of peroxisome proliferator-activated receptor γ（ PPARγ）,E-cadherin and vimentin were measured by immunohistochemistry. miR-130 b inhibitor synthesized artificially was transfected into SMMC-7721 cells in vitro. Cell invasion was analyzed by TranswellTMassay. The expressions of PPARγ,E-cardhern and vimentin in SMMC-7721 cells after transfected with miR-130 b inhibitor and PPARγ siRNA were detected by qRT-PCR and Western blotting. Results The expression of miR-130 b mRNA in HCC tissues was significantly higher than that in matched normal tumor-adjacent tissues. Clinical analysis indicated that high expression of miR-130 b was prominently correlated with venous infiltration,high Edmondson-Steiner grading and advanced tumor node metastasis（ TNM）stage. Elevated miR-130 b expression was observed in all HCC cell lines（ Hep G2,SMMC-7721,Huh7 and Hep3B） as compared with that in LO2 nontransformed hepatic cell line. Furthermore,there was an inverse correlation between miR-130 b and E-cadherin as well as between miR-130 b and PPARγ,and a positive correlation between miR-130 b and vimentin was demonstrated in HCC tissues. miR-130 b inhibitor could significantly increase the expression of PPARγ and E-cadherin,but decrease the expression of vimentin in SMMC-7721 cells,meanwhile it suppressed the migration and invasion of SMMC-7721 cells. In addition,the down-regulation of PPARγ expression by PPARγ siRNA partially abrogated the above effect of miR-130 b on HCC cells. Conclusion The expression of miR-130 b in HCC tissues is significantly higher than that in tumor-adjacent tissues. The increased expression of miR-130 b is related with the malignant manifestations of HCC. miR-130 b may promote HCC cell invasion by inhibiting PPARγ expression and inducing epithelial-mesenchymal transition.