miR-424-5p/Rictor/mTORC信号轴诱导肝癌细胞自噬性死亡的机制  

Mechanism of miR-424-5p/Rictor/mTORC signal axis inducing autophagic death of hepatocellular carcinoma cells

作　　者：梁辉 杨金平 范银白 LIANG Hui;YANG Jinping;FAN Yinbai(Department of Hepatobiliary and Pancreatic Surgery,the Second Affiliated Hospital of Nanchang University,Jiangxi Province,Nanchang330006,China;Department of Anesthesiology,Ganzhou Municipal Hospital,Jiangxi Province,Ganzhou341099,China;Department of Physical Examination,Ganzhou City People′s Hospital,Jiangxi Province,Ganzhou341000,China)

机构地区：[1]南昌大学第二附属医院肝胆胰腺外科,江西南昌330006 [2]江西省赣州市立医院麻醉科,江西赣州341099 [3]江西省赣州市人民医院体检科,江西赣州341000

出　　处：《中国当代医药》2022年第29期5-10,14,共7页China Modern Medicine

基　　金：江西省卫生健康委科技计划项目(20204269)。

摘　　要：目的研究miR-424-5p/Rictor/哺乳动物雷帕霉素靶蛋白复合物(mTORC)信号轴诱导肝癌细胞自噬性死亡的机制。方法选取肝癌细胞系Hep-3B,对照组经PBS处理,过表达miR-424-5p组、干扰miR-424-5p表达组、过表达Rictor组、干扰Rictor表达组分别转染miR-424-5p mimics、miR-424-5p inhibitor、Rictor过表达质粒、Rictor siRNA。检测细胞活性、凋亡、周期,观察切片自噬体形,检测细胞Lc3-Ⅱ、Lc3-Ⅰ、Beclin1、P62 mRNA和蛋白,RT-PCR测定miR-424-5p/Rictor/mTORC2/Akt/mTORC1的mRNA表达,Western blot法检测相关蛋白,并验证miR-424-5p与Rictor靶向关系。结果Hep-3B细胞中miR-424-5p低表达,表达量为(0.45±0.05)。转染后过表达miR-424-5p组G0+G1期细胞高于对照组,细胞活性低于对照组,差异有统计学意义(P<0.05)。干扰miR-424-5p表达组及过表达Rictor组G_(0)+G_(1)期细胞比例低于过表达miR-424-5p组,细胞活性高于过表达miR-424-5p组、干扰Rictor表达组,差异有统计学意义(P<0.05)。过表达miR-424-5p组在488、587 nm激发波长下自噬小体、自噬溶酶体均高于对照组。过表达miR-424-5p组Rictor、mTORC2、Akt、mTORC1表达水平低于干扰miR-424-5p表达组及过表达Rictor组,高于干扰Rictor表达组,差异有统计学意义(P<0.05)。过表达miR-424-5p组Lc3-Ⅱ、Lc3-Ⅰ、Beclin1表达水平低于另外四组,P62表达水平高于其他四组,差异有统计学意义(P<0.05)。双荧光素酶实验证实miR-424-5p可直接抑制Rictor表达,Western blot显示miR-424-5p可抑制Hep-3B细胞内Rictor表达。结论miR-424-5p可通过调控Rictor/mTORC2/Akt/mTORC1表达影响自噬,从而抑制肝癌细胞增殖,促进其凋亡。Objective To study the mechanism of miR-424-5p/Rictor/mammalian target of rapamycin(mTORC)signaling axis inducing autophagic death of hepatoma cells.Methods Hep-3B was selected as liver cancer cell,the control group was treated with PBS,and the overexpression miR-424-5p group,interference miR-424-5p expression group,overexpression Rictor group and interference Rictor expression group were respectively transfected with miR-424-5p mimics,miR-424-5p inhibitor,Rictor overexpression plasmid,Rictor siRNA.Cell viability,apoptosis and cycle were detected,the shape of autophagosomes in slices was observed,Lc3-Ⅱ,Lc3-Ⅰ,Beclin1,P62 mRNA and protein were detected in cells,and the mRNA expression of miR-424-5p/Rictor/mTORC2/Akt/mTORC1 was determined by RT-PCR,Western blot method was used to detect related proteins,and to verify the targeting relationship between miR-424-5p and Rictor.Results The expression of miR-424-5p in Hep-3B cells was low(0.45±0.05).After transfection,the G0+G1 phase cells in the overexpression miR-424-5p group were higher than those in the control group,and the cell viability was lower than that in the control group,the differences were statistically significant(P<0.05).The proportion of cells in G_(0)+G_(1) phase in the interference miR-424-5p expression group and overexpression Rictor group were lower than those in the overexpression miR-424-5p group,and the cell viability was higher than that in the overexpression miR-424-5p group,interference Rictor expression group,the differences were statistically significant(P<0.05).The autophagosomes and autophagolysosomes in the overexpression miR-424-5p group at excitation wavelengths of 488 nm and 587 nm were higher than those in the interference miR-424-5p expression group and overexpression Rictor group,the differences were statistically significant(P<0.05).The expression levels of Rictor,mTORC2,Akt and mTORC1 in the overexpression miR-424-5p group were lower than those in the interference miR-424-5p expression group and the overexpression Rictor group

关 键 词：miR-424-5p RICTOR 哺乳动物雷帕霉素靶蛋白复合物 免疫荧光 

分 类 号：R735.7[医药卫生—肿瘤]