过表达miR-22-3p抑制肝癌Li-7细胞增殖、迁移和上皮间质转化

Overexpression of miR-22-3p inhibits liver cancer Li-7 cell proliferation,migration,and epithelial-mesenchymal transition

目的探讨miR-22-3p对肝癌Li-7细胞增殖、迁移及上皮间质化转变的影响。方法应用脂质体3000将miR-22-3p过表达质粒(GV251/miR-22-3p)转染至肝癌Li-7细胞,实验分别设置正常对照组(N)、阴性对照组(GV251/NC)和miR-22-3p过表达组(GV251/miR-22-3p)。应用浓度为200μg/mL G418筛选出miR-22-3p过表达稳转细胞株。CCK8实验检测细胞增殖能力变化;细胞划痕实验检测细胞迁移能力变化;Real-time PCR和Western blot检测Bcl-2和Bax的表达以及上皮间质转化(epithelial-mesenchymal transition,EMT)相关标志基因(E-cadherin、β-catenin、N-cadherin和Vimentin)mRNA和蛋白表达。结果与N组和GV251/NC组相比,GV251/miR-22-3p组miR-22-3p的表达显著升高(P<0.01),而且细胞的增殖和迁移能力在48h后均显著降低(P<0.01,P<0.01);与N组和GV251/NC组相比,GV251/miR-22-3p组Bcl-2的表达显著降低(P<0.01),Bax的表达则明显升高(P<0.01);同时,上皮标志基因E-cadherin和β-catenin则明显升高(P<0.01),间质标志基因N-cadherin和Vimentin在mRNA和蛋白表达水平均显著降低(P<0.01)。结论miR-22-3p可通过抑制Bcl-2及促进Bax表达,进而降低肝癌Li-7细胞的增殖能力。同时,通过调控EMT相关基因的表达抑制EMT进程,进而降低肝癌Li-7细胞的迁移能力。

Objective To investigate the effects of miR-22-3p on the proliferation,migration and epithelial-mesenchymal transition(EMT)of liver cancer Li-7 cells.Methods The liposome 3000 was used to transfect the miR-22-3p over-expressing plasmid(GV251/miR-22-3p)into liver cancer Li-7 cells.The experiments were set up in normal group(N),negative control group(NC)and miR-22-3p over-expression group(GV251/miR-22-3p).The miR-22-3p stably transfected cell lines were selected using 200μg/mL G418.The CCK8 test was used to detect cell proliferation ability,the cell scratch test was used to detect cell migration ability,and Real-time PCR and Western blot were used to detect the expression of Bcl-2 and Bax,and EMT markers of E-cadherin,β-catenin,N-cadherin and Vimentin.Results Compared with the GV251/NC group,the expression of miR-22-3p in the GV251/miR-22-3p group was significantly increased(P<0.01),and the cell proliferation and migration ability were significantly reduced after 48 hours(P<0.01).Compared with N group and GV251/NC group,the expression of Bcl-2 in GV251/miR-22-3p group was significantly reduced(P<0.01),while the expression of Bax was significantly increased(P<0.01).Meanwhile,the EMT marker genes E-cadherin andβ-catenin increased significantly(P<0.01),while interstitial marker genes N-cadherin and Vimentin significantly reduced(P<0.01).Conclusion miR-22-3p can inhibit the proliferation of Li-7 cells by inhibiting Bcl-2 and promoting Bax expression.At the same time,miR-22-3p inhibits the EMT process by regulating the expression of EMT-related genes,thereby reducing the migration capacity of liver cancer Li-7 cells.