miR-124-3p靶向Beclin 1介导的自噬增强胃肠道间质瘤细胞对伊马替尼的敏感性  

miR-124-3p enhances sensitivity of gastrointestinal stromal tumors cells to imatinib by targeting Beclin 1-mediated autophagy

作　　者：胡珊珊[1] 赵海明[1] HU Shanshan;ZHAO Haiming(Department of Gastroenterology,Sichuan Provincial People's Hospital,School of Medicine,University of Electronic Science and Technology of China,Chengdu 610000,Sichuan,China)

机构地区：[1]四川省医学科学院·四川省人民医院(电子科技大学附属医院)消化内科,成都610000

出　　处：《医学研究与战创伤救治》2024年第4期337-345,共9页Journal of Medical Research & Combat Trauma Care

基　　金：四川省医学科研课题(S18082)。

摘　　要：目的旨在探讨微小RNA miR-124-3p与胃肠道间质瘤(GIST)细胞对伊马替尼(IM)敏感性的关系,并初步探讨可能的潜在机制。方法根据相应处理方式对GIST细胞进行分组:IM+GIST-T1组、IM+GIST-882组、miR-NC组、miR-124-3p mimic组、miR-124-3p inhibitor组、NC组、IM+miR-NC组、IM+miR-124-3p mimic组、IM+miR-124-3p inhibitor组、IM+miR-124-3p mimic+RAPA组、oe-NC组、oe-BECN1组、oe-BECN1+miR-124-3p mimic组。采用实时定量逆转录聚合酶链反应(qRT-PCR)检测细胞中miR-124-3p和Beclin-1(BECN1)mRNA的表达。采用细胞计数试剂盒-8测定法和EdU荧光染色法检测细胞活力和增殖。采用流式细胞术检测细胞凋亡。采用蛋白免疫印迹检测蛋白表达。采用免疫荧光染色检测自噬体形成。采用双荧光素酶报告基因实验验证miR-124-3p和BECN1的关系。采用皮下接种法构建GIST-T1细胞移植瘤裸鼠动物模型。结果qRT-PCR显示,与miR-NC组(0.023±0.002)相比,miR-124-3p mimic组(0.351±0.014)细胞中miR-124-3p表达显著升高(P<0.01),而miR-124-3p inhibitor(0.011±0.002)中miR-124-3p表达显著降低(P<0.01)。流式细胞术显示,转染miR-124-3p mimic可显著促进经IM处理的GIST-T1细胞的凋亡(P<0.01),而转染miR-124-3 inhibitor则会抑制细胞的凋亡(P=0.004)。CCK-8结果显示,RAPA处理能够在一定程度上逆转miR-124-3p对IM敏感性的增强作用。蛋白免疫印迹分析显示,与NC组相比,IM+miR-NC组细胞中BECN1蛋白表达和LC3II/LC3I比值显著增加,而p62蛋白表达显著降低(P<0.01)。与IM+miR-NC组相比,IM+miR-124-3p mimic组细胞中BECN1蛋白表达和LC3II/LC3I比值明显降低,而p62蛋白表达明显增加(P<0.01)。与IM+miR-124-3p mimic组相比,IM+miR-124-3p mimic+RAPA组BECN1蛋白表达和LC3II/LC3I比值显著增加,而p62蛋白表达显著降低(P<0.01)。流式细胞术结果显示,与NC组相比,IM+miR-NC组细胞凋亡率明显增加(P<0.01);与IM+miR-NC组相比,IM+miR-124-3p mimic组(细胞凋亡明显增加(P<0.01);与IM+Objective The aim of this study was to investigate the relationship between the microRNA miR-124-3p and the sensitivity of gastrointestinal stromal tumors(GIST)cells to imatinib(IM),and to preliminarily explore the possible underlying mechanisms.Methods According to the corresponding treatments,GIST cells were divided into im+GIST-T1 group,im+GIST-882 group,miR-NC group,miR-124-3p mimic group,miR-124-3p inhibitor group,NC group,im+miR-NC group,im+miR-124-3p mimic group,im+miR-124-3p inhibitor group,IM+miR-124-3p mimic+RAPA group,oe-NC group,oe-BECN1 group,oe-BECN1+miR-124-3p mimic group.Real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR)was used to detect the expression of miR-124-3p and Beclin-1(BECN1)mRNA in cells.Cell viability and proliferation were detected using cell counting kit-8 assay and EdU fluorescent staining.Apoptosis was detected using flow cytometry.Protein immunoblotting was used to detect protein expression.Autophagosome formation was detected using immunofluorescence staining.Dual luciferase reporter gene assay was used to verify the relationship between miR-124-3p and BECN1.The subcutaneous inoculation method was used to construct the GIST-T1 cell transplantation tumor nude mouse animal model.Results qRT-PCR showed that miR-124-3p expression was significantly elevated(P<0.01)in cells of miR-124-3p mimic group(0.351±0.014)compared to miR-NC group(0.023±0.002),while miR-124-3p inhibitor(0.011±0.002)had significantly downregulated miR-124-3p expression(P<0.01).Flow cytometry showed that transfection of miR-124-3p mimic significantly promoted apoptosis in IM-treated GIST-T1 cells(P<0.01),whereas transfection of miR-124-3 inhibitor inhibited apoptosis(P=0.004).CCK-8 results showed that RAPA treatment was able to reverse to some extent the miR-124-3p's enhancing effect on IM sensitivity.Protein immunoblotting analysis showed that BECN1 protein expression and LC3II/LC3I ratio were significantly increased,while p62 protein expression was significantly decreased in cells

关 键 词：胃肠道间质瘤 伊马替尼 微小RNA 自噬 

分 类 号：R735.3[医药卫生—肿瘤]