血清外泌体介导lncRNA HOTTIP通过miR-138-5p/TJP1轴调控胃癌细胞顺铂耐药的研究  

Serum exosome-mediated lncRNA HOTTIP regulates cisplatin resistance in gastric cancer cells through the miR-138-5p/TJP1 axis

作　　者：韩明阳[1] 白军伟[1] 聂洁伟[1] 李媛媛[2] 张超[1] Han Mingyang;Bai Junwei;Nie Jiewei;Li Yuanyuan;Zhang Chao(Department of Gastrointestinal Surgery,Henan Provincial People′s Hospital,Zhengzhou 450003,China;Reproductive Hospital,Henan Provincial People′s Hospital,Zhengzhou 450003,China)

机构地区：[1]河南省人民医院胃肠外科,郑州450003 [2]河南省人民医院生殖医院,郑州450003

出　　处：《中华消化外科杂志》2023年第12期1467-1475,共9页Chinese Journal of Digestive Surgery

基　　金：河南省医学科技攻关项目(SB201902025)。

摘　　要：目的探讨胃癌患者血清外泌体对胃癌细胞AGS顺铂耐药、克隆形成、迁移、侵袭和凋亡的影响,并探讨其分子机制。方法分离胃癌患者血清外泌体,qRT-PCR检测血清外泌体中lncRNA HOTTIP的表达。体外培养正常胃上皮细胞株GES1、胃癌细胞株AGS、人胚肾细胞株293T。AGS细胞与外泌体(Exo)孵育,以PBS处理作为对照,随后转染si-NC或si-HOTTIP-3,设为Exo组、PBS组、si-NC+Exo组、si-HOTTIP-3+Exo组。AGS细胞分别转染si-NC、si-HOTTIP-1、si-HOTTIP-2、si-HOTTIP-3、oe-HOTTIP、vector、oe-HOTTIP+miR-138-5p mimic、oe-HOTTIP+mimic NC、miR-138-5p inhibitor、inhibitor NC、miR-138-5p inhibitor+si-TJP1及miR-138-5p inhibitor+si-NC,设为si-NC组、si-HOTTIP-1组、si-HOTTIP-2组、si-HOTTIP-3组、oe-HOTTIP组、vector组、oe-HOTTIP+miR-138-5p mimic组、oe-HOTTIP+mimic NC组、miR-138-5p inhibitor组、inhibitor NC组、miR-138-5p inhibitor+si-TJP1组及miR-138-5p inhibitor+si-NC组。293T细胞转染mimic NC+HOTTIP wt、miR-138-5p mimic+HOTTIP wt、mimic NC+HOTTIP mut、miR-138-5p mimic+HOTTIP mut、mimic NC+TJP1 3′UTR wt、miR-138-5p mimic+TJP1 3′UTR wt、mimic NC+TJP1 3′UTR mut、miR-138-5p mimic+TJP1 3′UTR mut分别设为mimic NC+HOTTIP wt组、miR-138-5p mimic+HOTTIP wt组、mimic NC+HOTTIP mut组、miR-138-5p mimic+HOTTIP mut组、mimic NC+TJP1 3′UTR wt组、miR-138-5p mimic+TJP1 3′UTR wt组、mimic NC+TJP1 3′UTR mut组、miR-138-5p mimic+TJP1 3′UTR mut组。CCK8实验检测细胞的顺铂敏感性,平板克隆检测细胞克隆形成,Transwell实验检测细胞迁移和侵袭,流式细胞仪检测细胞凋亡,Western blot实验检测外泌体标志蛋白CD63和CD81、TJP1及耐药相关蛋白P-gp、MCL-1的表达,双荧光素酶实验验证lncRNA HOTTIP、miR-138-5p和TJP1之间的靶向关系。观察指标:(1)lncRNA HOTTIP的表达情况。(2)血清外泌体介导lncRNA HOTTIP对胃癌细胞顺铂的耐药情况。(3)过表达lncRNA HOTTIP通过miR-138-5p对胃癌细胞顺铂耐药的调控情况。(4)miR-Objective To investigate the effects of serum exosomes of patients with gastric cancer on cisplatin resistance,clonal formation,migration,invasion and apoptosis of the AGS gastric cancer cells,and the corresponding molecular mechanisms.Methods The experimental study was conducted.The exosomes of patients with gastric cancer was separated from their serum,and the expression of lncRNA HOTTIP was analyzed using the quantitative real time polymerase chain reaction(qRT-PCR).Normal gastric epithelial cell line GES1,gastric cancer cell line AGS and human embryonic kidney cell 293T were cultured in vitro.AGS cells were incubated with exosomes(Exo),with phosphate buffered saline(PBS)treatment as control,and transfected with si-NC or si-HOTTIP-3,named as Exo group,PBS group,si-NC+Exo group,and si-HOTTIP-3+Exo group.The AGS cells were transfected with si-NC,si-HOTTIP-1,si-HOTTIP-2,si-HOTTIP-3,oe-HOTTIP,vector,oe-HOTTIP+miR-138-5p mimic,oe-HOTTIP+mimic NC,miR-138-5p inhibitor,inhibitor NC,miR-138-5p inhibitor+si-TJP1 and miR-138-5p inhibitor+si-NC.They were recorded as si-NC group,si-HOTTIP-1 group,si-HOTTIP-2 group,si-HOTTIP-3 group,oe-HOTTIP group,vector group,oe-HOTTIP+miR-138-5p mimic group,oe-HOTTIP+mimic NC group,miR-138-5p inhibitor group,inhibitor NC group,miR-138-5p inhibitor+si-TJP1 group and miR-138-5p inhibitor+si-NC group.The 293T cells transfected with mimic NC+HOTTIP wt,miR-138-5p mimic+HOTTIP wt,mimic NC+HOTTIP mut,miR-138-5p mimic+HOTTIP mut,mimic NC+TJP1 3′UTR wt,miR-138-5p mimic+TJP1 3′UTR wt,mimic NC+TJP1 3′UTR mut,miR-138-5p mimic+TJP1 3′UTR mut were recorded as the mimic NC+HOTTIP wt group,miR-138-5p mimic+HOTTIP wt group,mimic NC+HOTTIP mut group,miR-138-5p mimic+HOTTIP mut group,mimic NC+TJP1 3′UTR wt group,miR-138-5p mimic+TJP1 3′UTR wt group,mimic NC+TJP1 3′UTR mut group,miR-138-5p mimic+TJP1 3′UTR mut group.The cell counting kit-8(CCK8)was used to analyze the cisplatin sensitivity of gastric cancer cells.The colony formation experiment was used to analyze the colony formation of gas

关 键 词：胃肿瘤 血清外泌体 lncRNA HOTTIP miR-138-5p TJP1 顺铂耐药 

分 类 号：R735.2[医药卫生—肿瘤]