miR-543调控ATG-7影响肝癌细胞HepG2凋亡  

miR-543 effect cell apoptosis of HepG2 through its target gene ATG-7

作　　者：余涛 吴琪芳 YU Tao;WU Qifang(The Affiliated Hospital of Jiujiang Vniversity in Jiangxi province,Jiujiang 332000)

机构地区：[1]江西省九江学院附属医院,九江332000

出　　处：《江西医药》2023年第3期271-276,共6页Jiangxi Medical Journal

摘　　要：目的探讨microRNA-543(miR-543)通过调控其靶基因自噬相关基因7(ATG-7)对肝癌细胞凋亡的影响。方法通过荧光定量PCR及western blot实验观察多株肝癌细胞株里miR-543和ATG-7的表达情况;选取与人源胚胎肾细胞(293T)相比ATG-7高表达的HepG2肝癌细胞分组,瞬时转染miR-543摸拟物和miR-543抑制剂,在HepG2细胞中使用Western blot方法检测ATG-7蛋白表达变化,再使用qRT-PCR检测miR-543和ATG-7的mRNA水平;经过microRNA数据库预测miR-543与ATG-7的结合区域,构建ATG-73’UTR野生型及突变型的荧光素酶质粒和miR-543质粒后,通过荧光素酶报告基因实验观察野生型及突变型的荧光素酶活性变化;用流式细胞仪检测转染miR-543模拟物和ATG-7质粒的HepG2细胞凋亡情况。结果多株肝癌细胞株中ATG-7蛋白水平表达情况不一,ATG-7的mRNA和miR-543表达水平呈负相关;选用ATG-7相对高表达的HepG2细胞,过表达miR-543后发现miR-543 mRNA升高后,ATG-7蛋白和mRNA水平随之降低;降低miR-543,ATG-7蛋白和mRNA水平随之升高;通过荧光素酶报告基因证实miR-543结合ATG-7 mRNA 3’UTR区域的特定序列来降低ATG-7的mRNA水平;通过细胞凋亡实验证实miR-543通过下调ATG-7抑制肝癌细胞凋亡。结论在肝癌细胞中miR-543下调ATG-7抑制肝癌细胞的凋亡。Objective In order to study the function of microRNA-543(miR-543)on cell apoptosis of hepatocellular carcinoma through regulating the autophagy related gene 7(ATG-7).Methods To know the expression levels of miR-543 and ATG-7 in multiple cell lines of hepatocellular carcinoma by qPCR and western blotting,and then select hepatocellular carcinoma cell line HepG2 with the highest expression of ATG-7 compared with human embryonic kidney cells(293T),to observe the expression change of ATG-7 protein in HepG2 cells by Western blot after transfected miR-543 mimic and miR-543 inhibitor,and again to observe miR-543 and ATG-7 mRNA levels through the qRT-PCR;after predicting the combined ATG-7 region of miR-543 with microRNA database,building ATG-73"UTR wild type and mutant luciferase plasmid,miR-543 plasmid,and then to observe luciferase activity changes of wild type and mutant.After infected by miR-543 mimic and ATG-7 plasmid,HepG2 cell apoptosis was analyzed by flow cytometry.Results n many hepatoma cell lines,ATG-7 protein expression levels were different and ATG-7 and miR-543 mRNA levels was negatively correlated.HepG2 cell was selected because of ATG-7 relatively highest expression.After over-expression of miR-543 where miR-543 mRNA was increased,ATG-7 protein and mRNA levels decreased.And after inhibiting miR-543,ATG-7 protein and mRNA levels increased.Luciferase gene reporters confirmed that miR-543 reduced ATG-7 mRNA level by binding the specific sequence of ATG-7 mRNA 3'UTR regio.Cell apoptosis experiment showed that miR-543 inhibited cell apoptosis of HCC through down-regulating ATG-7.Conclusion miR-543 can inhibit cell apoptosis of hepatocellular carcinoma through down-regulating ATG-7,that is to say,miR-543 may be the cancer gene in hepatocellular carcinoma.

关 键 词：自噬相关基因7(ATG-7) 肝癌 细胞凋亡 

分 类 号：R735.7[医药卫生—肿瘤]