乙酰肝素酶通过ERK/MMP⁃9信号通路促进胆囊癌细胞侵袭和迁移

Heparanase promotes the invasion and migration of gallbladder cancer cells via ERK/MMP⁃9 signaling pathway

目的研究乙酰肝素酶(HPSE)对胆囊癌细胞侵袭迁移以及ERK/MMP⁃9信号通路作用机制。方法在胆囊癌细胞(GBC⁃SD)中采用慢病毒转染的方法双向调节HPSE基因的表达,获得转基因过表达HPSE的胆囊癌细胞株和HPSE极低表达的胆囊癌细胞株,qRT⁃PCR和Western blot检测过表达及干扰效果。利用Transwell及细胞划痕实验分别检测转染后阴性对照组、HPSE敲低组及过表达组胆囊癌细胞侵袭及迁移能力。同时,qRT⁃PCR检测HPSE、MMP⁃9 mRNA转录水平,Western blot检测MMP⁃9、HPSE、p⁃ERK、ERK、N⁃cadherin及E⁃cadherin蛋白表达水平。结果HPSE基因过表达组和干扰组胆囊癌细胞株成功构建(P<0.05)。过表达HPSE显著促进胆囊癌细胞的迁移及侵袭,同时MMP⁃9 mRNA和蛋白表达及p⁃ERK、N⁃cadherin蛋白表达均上调,E⁃cadherin表达下调;敲低HPSE表达后情况相反(P<0.05)。另外,过表达HPSE与ERK抑制剂共同处理逆转了HPSE基因过表达对胆囊癌细胞生物学功能的促进作用(P<0.05)。结论乙酰肝素酶通过激活ERK信号通路上调MMP⁃9增强胆囊癌细胞的侵袭迁移能力。

Objective To investigate the effect of heparanase(HPSE)on invasion and migration of gall⁃bladder cancer cells and ERK/MMP⁃9 signaling pathway.Methods Lentivirus transfection was used to bidirectional regulate the expression of HPSE gene in gallbladder cancer cells(GBC⁃SD)to obtain a transgenic gallbladder cancer cell line with overexpression of HPSE and a gallbladder cancer cell line with very low expression of HPSE.The overexpression and interference effects of HPSE were detected by qRT⁃PCR and Western blot.Transwell and cell scratch test were used to detect the invasion and migration ability of gallbladder cancer cells in negative control group,HPSE knockdown group and HPSE overexpression group before and after transfection.At the same time,the mRNA transcription levels of HPSE and MMP⁃9 were detected by qRT⁃PCR,and the protein expression levels of MMP⁃9,HPSE,p⁃ERK,ERK,N⁃cadherin and E⁃cadherin were detected by Western blot.Results The gallbladder cancer cell lines of HPSE gene overexpression group and interference group were successfully constructed(P<0.05).Overexpression of HPSE significantly promoted the migration and invasion of gallbladder cancer cells,and the expression of MMP⁃9 mRNA and protein,p⁃ERK and N⁃cadherin protein was up⁃regulated,while the expression of E⁃cadherin was down⁃regulated.After knockdown of HPSE expression,the situation was opposite(P<0.05).In addition,the co⁃treatment of HPSE overexpression and ERK inhibitor reversed the promotion effect of HPSE gene overexpression on the biological function of gallbladder cancer cells(P<0.05).Conclusions Heparanase enhances the invasion and migration of gallbladder cancer cells by activating ERK signaling pathway and upregulating MMP⁃9.