过表达Cx32对肝细胞癌细胞Huh7增殖、迁移、侵袭能力的影响及其机制

Effect of Cx32 over-expression on cell proliferation,migration,and invasion of hepatocellular carcinoma cell line Huh7 and its mechanism

目的:揭示缝隙连接蛋白32(connexin 32,Cx32)对肝细胞癌(heptocellular carcinoma,HCC)细胞Huh7增殖、迁移、侵袭的影响及其机制。方法:首先,利用生物信息学技术分析Cx32在癌组织与正常肝组织中的表达差异及其与HCC重要临床病理特征之间的关系;其次,在细胞水平检测Cx32的表达在4种HCC细胞株与正常肝脏上皮细胞株中的差异,构建稳定过表达Cx32的Huh7细胞株,并采用MTT法、细胞划痕实验和Transwell实验检测过表达Cx32对Huh7细胞增殖、迁移及侵袭能力的影响;最后,使用蛋白质免疫印迹法(westernblot)和免疫荧光法(immunofluorescence,IF)检测上皮-间质转化(epithelialmesenchymal transition,EMT)标志物表达水平的变化。结果:生信分析显示,相较于正常肝组织,Cx32mRNA和蛋白质表达水平在HCC组织中降低,且mRNA表达水平随着HCC患者T分期、组织学分级及临床分期的进展而降低(P<0.05)。细胞实验结果示,Cx32蛋白表达在不同HCC细胞株中均呈现一定程度的下调;其中,经慢病毒感染介导Cx32稳定高表达的Huh7细胞(Cx32 OE)与空白对照(Control)组和(或)阴性对照(NC)组细胞相比,OD490值、划痕愈合率及侵袭细胞数均更低(P<0.05)。Cx32 OE组的E-cadherin蛋白表达水平上调,而Snail和Vimentin蛋白表达水平下调。结论:Cx32在HCC组织和细胞中均低表达,而Huh7细胞的增殖、迁移和侵袭能力可被过表达的Cx32抑制,Cx32这一效应可能与其对EMT过程的抑制有关。

Objective:To explore the effect of connexin 32(Cx32)on the cell proliferation,migration,invasion of human hepatocellular carcinoma(HCC)cell line Huh7 and its mechanism.Methods:Firstly,bioinformatics techniques were used to analyze the difference in expression of Cx32 between HCC tissues and normal liver tissues,and the relationship between Cx32 expression and important clinicopathological features of HCC was also explored.Subsequently,Cx32 expression in HCC cell lines and normal hepatic epithelial cell line was detected in vitro.Huh7 cell line with stable over-expression of Cx32 was further established,and the change in cell proliferation ability was measured by MTT assay,changes in migration and invasion capacities were detected by wound-healing assay and transwell assay,on this cell line.Finally,western blot and immunofluorescence(IF)were used to investigate the alterations of expression of epithelial-mesenchymal transition(EMT)markers.Results:Bioinformatics analyses showed that Cx32 mRNA and protein expression levels in HCC tissues were lower than those in normal liver tissues,and the mRNA expression level of Cx32 was negatively correlated with T stage,histological grade and clinical stage of HCC patients(all P<0.05).Results of in vitro experiments revealed Cx32 protein expression in different HCC cell lines was down-regulated compared to that in normal hepatic epithelial cell line LO2.Cx32 stably over-expressed(Cx32 OE)Huh7 cell line was successfully constructed by lentivirus infection and showed high expression of Cx32 protein in the cell line.Compared to the control group and(or)the negative control(NC)group,the Cx32 OE group exhibited decreased OD490 value,wound healing rate and invasive cell number(all P<0.05).Furthermore,an increase in the expression of epithelial marker E-cadherin,and a decrease in the expression of mesenchymal markers Snail and Vimentin,were observed in Cx32-OE Huh7 cell line.Conclusion:Cx32 is low expressed in HCC tissues and cells,while the proliferation,migration and invasion ability of Huh7 cells can be inhibited by over-expression of Cx32,of which the underlying mechanism may be related to the inhibition of EMT process.