慢病毒介导Akt1 shRNA载体转染对人肝母细胞瘤细胞株HepG2凋亡和增殖的影响  

Effects of Lentivirus-Mediated Akt1 shRNA Vector Transfection on Apoptosis and Proliferation of Hepatoblasts HepG2 Cells

作　　者：马体栋[1] 赵凡[1] 李碧香[1] MA Tidong;ZHAO Fan;LI Bixiang(Department of Neonatal Surgery,Hunan Children’s Hospital,Changsha,Hunan,410007,China)

机构地区：[1]湖南省儿童医院新生儿外科

出　　处：《肿瘤药学》2019年第4期572-577,共6页Anti-Tumor Pharmacy

摘　　要：目的探究慢病毒介导Akt1 shRNA载体转染对人肝母细胞瘤细胞株HepG2凋亡和增殖水平的影响。方法以人肝母细胞瘤细胞株HepG2为研究对象,将其分为空白对照组(A组)、空白载体病毒组(B组)和ShRNA-Akt1慢病毒组(C组)。A组细胞未作任何处理,B组细胞转染空白载体慢病毒,C组细胞转染shRNA-Akt1载体构建的慢病毒。利用短发夹RNA(shRNA)干扰技术构建Akt1的基因下调载体,通过慢病毒转染HepG2细胞,qPCR和Western blotting检测凋亡相关因子Caspase-3和Caspase-9的表达;CCK-8法检测细胞增殖能力;Transwell法检测细胞侵袭能力;Annexin-V/PI法检测细胞凋亡水平。结果shRNA-Akt1载体慢病毒转染滴度为8×10^7 TU·mL^-1。三组细胞的Akt1 mRNA相对表达量比较,差异均具有统计学意义(F=9.904,P=0.025),且C组显著低于A组和B组(q=3.092,2.009;P=0.019);三组细胞的Caspase-3和Caspase-9 mRNA相对表达量比较,差异均具有统计学意义(P<0.05),且C组显著高于A组和B组(P<0.05)。Western blotting检测结果显示,三组细胞的Akt1蛋白水平比较,差异均具有统计学意义(F=11.002,P=0.011),且C组显著低于A组和B组(q=2.887,2.457;P=0.014);三组细胞的Caspase-3和Caspase-9蛋白水平比较,差异均具有统计学意义(P<0.05),且C组显著高于A组和B组(P<0.05)。CCK-8结果显示,C组细胞增殖率显著低于B组和A组(q=2.009,3.901;P<0.05)。Transwell结果显示,C组细胞的侵袭能力显著低于B组和A组(q=2.127,2.157;P<0.05)。Annexin-V/PI结果显示,C组细胞凋亡率显著高于B组和A组(q=2.445,2.659;P<0.05)。结论慢病毒介导Akt1 shRNA载体可抑制HepG2细胞增殖和侵袭,并促进其凋亡,可作为肝癌的潜在治疗靶点。Objective To investigate the effect of lentivirus-mediated transfection of Akt1 shRNA vector on apoptosis and proliferation of hepatoblasts HepG2.Methods HepG2 cells were divided into blank control group(group A),blank vector virus group(group B)and SHRNA-Akt1 lentivirus group(group C).Group A were normal cells without any treatment.Group B were cells transfected with lentivirus.And group C were cells transfected with lentivirus constructed by shRNA-Akt1 vector.The down-regulation vector of Akt1 gene was constructed by short hairpin RNA(shRNA)interference technique.Lentivirus was transfected into hepatoblasts HepG2 cells.Apoptosis-related factors Caspase-3 and Caspase-9 were detected by qPCR and Western blotting.The proliferation,invasive ability and apoptosis level of hepatoblasts HepG2 cells were detected by CCK-8,Transwell and Annexin-V/PI methods.Results The transfection titer of shRNA-Akt1 vector lentivirus was 8×10^7 TU·mL^-1.The relative mRNA expression of Akt1 in group A,B and C was significantly different(F=9.904,P=0.025),and it was significantly lower in group C than in group A and B(q=3.092,2.009;P=0.019).The differences between groups in mRNA expression of Caspase-3 and Caspase-9 were the same as that of Akt1.Western blotting results showed that there were significant differences in the protein levels of Akt1 between group A,B and C(F=11.002,P=0.011).The protein levels of Akt1 were significantly lower in group C than in group A and B(q=2.887,2.457,P=0.014),and so were the protein levels of Caspase-3 and Caspase-9.CCK-8 showed that the cell proliferation rate in group C was significantly lower than in group B and group A(q=2.009,3.901;P<0.05).Transwell results showed that the invasive ability of cells in group C was significantly lower than in group B and group A(q=2.127,2.157;P<0.05).Annexin-V/PI results showed that the apoptotic rate in group C was significantly higher than in group B and group A(q=2.445,2.659;P<0.05).Conclusion Lentivirus-mediated Akt1 shRNA vector could inhibit the proliferation

关 键 词：AKT1 HEPG2细胞 侵袭 增殖 凋亡 

分 类 号：R735.7[医药卫生—肿瘤]