miR-30a-5p靶向JAK1调控人胃腺癌AGS细胞自噬的机制研究  

Mechanism of miR-30a-5p Targeting JAK1 to Regulate Autophagy in Human Gastric Adenocarcinoma AGS Cells

作　　者：刘海英 曹天柱 崔自重 Liu Haiying;Cao Tianzhu;Cui Zizhong(Department of Interventional Vascular Surgery,Room 24,General Surgery Department,Department of Hematology and Oncology,The Third Hospital of Changsha,Changsha,Hunan 410015,China)

机构地区：[1]长沙市第三医院介入血管科,普通外科二十四病室,血液肿瘤科,湖南长沙410015

出　　处：《四川医学》2024年第7期709-715,共7页Sichuan Medical Journal

基　　金：长沙市自然科学基金(编号:kq2202014)。

摘　　要：目的探究miR-30a-5p靶向JAK1调控人胃腺癌AGS细胞自噬的机制。方法体外培养人正常胃黏膜上皮细胞(GES-1)和胃腺癌细胞(AGS)。RT-qPCR检测miR-30a-5p表达水平,Starbase数据库预测miR-30a-5p的表达水平及其与JAK1的潜在结合位点,双荧光素酶报告实验验证miR-30a-5p对JAK1基因的靶向调控作用,免疫荧光检测自噬标志物LC3阳性细胞水平,WB检测JAK1和自噬相关蛋白(Beclin-1、LC3 Ⅱ/Ⅰ和p62)的表达水平。结果与GES-1组相比,AGS组细胞中miR-30a-5p表达水平显著降低;过表达miR-30a-5p后,AGS细胞中自噬相关蛋白(Beclin-1、LC3 Ⅱ/Ⅰ)表达水平以及自噬标志物LC3阳性细胞水平显著降低,p62蛋白的表达水平显著升高;miR-30a-5p与JAK1存在结合位点且靶向负调控JAK1;与miR-30a-5p过表达+oe-NC对照处理,miR-30a-5p过表达同时过表达JAK1处理后观察到AGS细胞中自噬相关蛋白Beclin-1、LC3Ⅱ/Ⅰ的蛋白表达水平明显增加,p62蛋白水平明显降低,进一步的免疫荧光也观察到miR-30a-5p过表达+过表达JAK1后LC3荧光强度明显增加。结论miR-30a-5p靶向负调控JAK1蛋白的表达,抑制人胃腺癌AGS细胞自噬。Objective To explore the mechanism by which miR-30a-5p targets JAK1 to regulate autophagy in human gastric adenocarcinoma AGS cells.Methods Human normal gastric mucosal epithelial cells(GES-1)and gastric adenocarcinoma cells(AGS)were cultured in vitro.RT-qPCR was performed to detect the expression level of miR-30a-5p.The Starbase database was used to predict the expression level of miR-30a-5p and its potential binding site to JAK1,and dual-luciferase reporter assay was performed to verify the target-regulatory effect of miR-30a-5p on JAK1.Immunofluorescence was conducted to detect the level of LC3-positive cells for autophagy markers,and WB was utilized to detect the expression level of JAK1 and autophagy-related proteins(Beclin-1,LC3 Ⅱ/Ⅰ,and p62).Results The expression level of miR-30a-5p in the AGS group was significantly lower than that in the GES-1 group,with significantly lower expression levels of autophagy-related proteins(Beclin-1 and LC3 Ⅱ/Ⅰ)and the autophagy marker LC3 and markedly higher expression levels of p62 protein.miR-30a-5p had a binding site to JAK1 and targeted JAK1 negatively.Finally,rescue experiments displayed that compared with the miR-30a-5p mimics+oe-NC group,the miR-30a-5p mimics+oe-JAK1 group exhibited substantial increases in the expression level of autophagy-related proteins(Beclin-1 and LC3 Ⅱ/Ⅰ)and significant reductions in p62 protein expression in AGS cells.Further immunofluorescence also revealed a significant increase in LC3 fluorescence intensity after miR-30a-5p overexpression plus JAK1 overexpression in AGS cells.Conclusion miR-30a-5p targets and negatively regulates JAK1 protein expression and inhibits autophagy in human gastric adenocarcinoma AGS cells.

关 键 词：胃腺癌 miR-30a-5p JAK1 AGS细胞 细胞自噬 

分 类 号：R730.23[医药卫生—肿瘤]