靶向Atg5的siRNAs促进肝癌细胞HCC-LM3失巢凋亡的实验研究  

Atg5-targeting siRNA promotes anoikis in hepatoma HCC-LM3 cells

作　　者：邓欢[1] 蔺朝妮 胡鹏[1] 王志毅[1] 罗敏[1] Deng Huan Lin Zhaoni Hu Peng Wang Zhiyi Luo Min(Department of Infection, Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China Second Department of lnternal Medicine, Chongqing Southeast Hospital, Chongqing 400060, China)

机构地区：[1]重庆医科大学附属第二医院感染科,重庆400010 [2]重庆市东南医院内二科,400060

出　　处：《现代医药卫生》2017年第15期2273-2276,共4页Journal of Modern Medicine & Health

摘　　要：目的研究靶向自噬相关基因5(Atg5)的小干扰RNA(siRNAs)能否诱导肝癌细胞的失巢凋亡,探讨细胞自噬是否参与肝癌细胞的失巢凋亡抵抗。方法选择高、低转移潜能的肝癌细胞HCC-LM3、SMMC-7721及人胚肝细胞LO_2,采用流式细胞术检测细胞在失巢培养状态下的凋亡率,采用蛋白质印迹(Western blotting)检测细胞Atg5蛋白表达。针对Atg5的siRNAs,采用脂质体转染法转染至高转移潜能的HCC-LM3肝癌细胞,利用Western blotting检测Atg5蛋白表达变化。在失巢状态下培养转染Atg5 siRNAs的肝癌细胞HCC-LM3,流式细胞术检测肝癌细胞的失巢凋亡率,Western blotting检测肝癌细胞中自噬小体膜蛋白LC3Ⅰ、Ⅱ型蛋白表达转变验证细胞自噬水平的变化。结果失巢培养后,HCC-LM3、SMMC-7721细胞失巢凋亡率明显低于人胚肝细胞LO_2,差异均有统计学意义(P<0.05);而在两组肿瘤细胞中,HCC-LM3细胞的失巢凋亡率低于SMMC-7721细胞,差异有统计学意义(P<0.05)。失巢培养后,HCC-LM3及SMMC-7721细胞LC3Ⅱ型蛋白表达均较LO_2细胞明显增加,差异均有统计学意义(P<0.05)。转染Atg5 siRNA1和siRNA2的肝癌HCC-LM3细胞中,Atg5蛋白的表达均明显低于阴性对照HCC-LM3-NC,差异均有统计学意义(P<0.05)。细胞中LC3Ⅱ型蛋白表达较阴性对照HCC-LM3-NC细胞明显减少,差异有统计学意义(P<0.05)。失巢凋亡率较阴性对照HCC-LM3-NC细胞明显增加,差异有统计学意义(P<0.05)。结论靶向Atg5的siRNAs能抑制肝癌细胞Atg5蛋白表达,并通过下调自噬水平来诱导肝癌细胞的失巢凋亡。Objective To investigate whether autophagy related gene 5（Atg5） targeting siRNAs could induce anoikis apoptosis in hepatocellular carcinoma cells,and to explore whether cell autophagy participating in the anoikis apoptosis resistance in hepatoma cells. Methods Hepatocellular carcinoma cells HCC-LM3 with high and low metastatic potential,SMMC-7721 and human embryo hepatocytes LO_2 were selected for this experiment. The apoptosis rate in anoikis culture status was detected by flow cytometry. The Atg5 protein expression change was detected by Western blot. Atg5 targeting siRNAs were transfected into hepatoma HCC-LM3 cells with high metastatic potential by liposome mediated transfection. The change of Atg5 protein expression was detected by Western blot. Hepatoma cells HCC-LM3 transfected with Atg5 siRNAs was cultured under the anoikis status,then the anoikis apoptosis rate of hepatoma cells was evaluated using flow cytometry. The change of cell autophagy level was con-firmed by LC3 Ⅰ,Ⅱ protein expression transformation detection via Western blot. Results After anoikis culture,the anoikis apoptosis rate of HCC-LM3 and SMMC-7721 cells were significantly lower than those of human embryo hepatocytes LO_2,the dif ferences were statistically significant（P〈0.05）;while in the two tumor cell groups,the anoikis apoptosis rate of HCC-LM3 cells was lower than that of SMMC-7721 cells,the difference was statistically significant（P〈0.05）. After anoikis culture,the LC3 typeⅡ protein expression in HCC-LM3 and SMMC-7721 cells were significantly increased compared with LO_2 cells,the differences were statistically significant（P〈0.05）. In hepatoma HCC-LM3 cells which transfected with Atg5 siRNA1 and siRNA2,Atg5 protein expression was significantly lower than that of negative control HCC-LM3-NC cells,the difference was statistically significant（P〈0.05）. The LC3 type Ⅱ protein expression in cells was significantly decreased compared with negative control HCC-LM3-NC cells,the difference was statisticall

关 键 词：肝细胞癌 失巢凋亡 自噬 自噬相关基因5 小干扰RNA 

分 类 号：R394[医药卫生—医学遗传学]