Hsa_circ_0000128通过miR-623/EIF4A1轴促进人胃腺癌细胞增殖和迁移的机制研究  

Mechanism of Hsa_circ_0000128/miR-623/EIF4A1 axis promoting proliferation and migration of human gastric adenocarcinoma cells

作　　者：王亚东 曹碧月 高生宝 王雪君 步雪峰[1] WANG Ya-dong;CAO Bi-yue;GAO Sheng-bao;WANG Xue-jun;BU Xue-feng(Department of General Surgery,the Affiliated People’s Hospital of Jiangsu University,Zhenjiang 212000,Jiangsu,China;Department of Respiratory,the Affiliated People’s Hospital of Jiangsu University,Zhenjiang 212000,Jiangsu,China)

机构地区：[1]江苏大学附属人民医院普外科,镇江212000 [2]江苏大学附属人民医院呼吸内科

出　　处：《医学研究生学报》2022年第10期1022-1029,共8页Journal of Medical Postgraduates

基　　金：国家自然科学基金(81672999);镇江市重点研发计划项目(SH20200034)。

摘　　要：目的目前对于hsa_circ_0000128是否通过miR-623/EIF4A1轴通路参与胃腺癌的发生过程尚不清楚。文中旨在研究环状RNA hsa_circ_0000128(Circ_01607)在胃腺癌组织、细胞株的表达以及对胃癌细胞的增殖、迁移、凋亡的影响。方法收集2019年9月至2020年8月江苏大学附属人民医院病理科的40份胃腺癌及癌旁组织标本。进行高通量测序筛选出表达明显提高的环状RNA hsa_circ_0000128;通过circBank、TargetScan等数据库预测下游miR-623。采用RT-PCR对40例临床胃腺癌组织、癌旁组织、胃癌细胞株(MGC-803,HGC-27,BGC-823)以及正常人胃粘膜上皮细胞株GES-1中hsa_circ_0000128的表达进行测定及验证结果。挑选出表达量差异最大的两组进行后续实验。将HGC-27、MGC-803细胞株分别接种于两块细胞培养板中,培养板中设置相应的circRNA组别:Si-NC组(Si-NC转染胃腺癌细胞)、Si-circ组(si-hsa_circ_0000128转染胃腺癌细胞)、Si-EIF4A1组(Si-EIF4A1转染胃腺癌细胞)、miR-NC组(miR-623-NC转染胃腺癌细胞)、miR-mimics组(miR-623-mimics转染胃腺癌细胞)、miR-inh组(miR-623-inhibitors转染胃腺癌细胞)、Si-circ+miR-NC组(Si-circ、miR-NC转染胃腺癌细胞)、Si-circ+miR-inh组(Si-circ、miR-inh转染胃腺癌细胞)。采用克隆形成实验测定各组细胞增殖能力;使用Transwell实验测定各组胃癌细胞的迁移能力;蛋白免疫印迹实验测定凋亡相关蛋白Cleaved-caspase-3、bcl-2和Bax的表达水平验证细胞凋亡情况。验证miR-623在组织及细胞中的表达情况,通过荧光素酶实验验证结合靶点信息,加入miRNA对应干扰并将其分组,通过细胞功能学实验,蛋白印迹实验比较胃癌细胞株各组细胞功能的变化。通过PCR及蛋白免疫印迹实验验证各组真核翻译起始因子4A1(EIF4A1)的相关表达水平与miR-623含量的关系。结果hsa_circ_0000128在胃癌组织及细胞株中较癌旁组织及正常胃黏膜上皮细胞中呈高表达状态。SI-circ组hsa_ciObjective At present,it is not clear whether hsa_circ_0000128 participates in the pathogenesis of gastric adenocarcinoma through miR-623/EIF4A1 axis.This study was aimed to investigate the expression of circRNA hsa_circ_0000128(Circ_01607)in gastric adenocarcinoma tissues and cell lines and its effect on the proliferation,migration and apoptosis of gastric cancer cells.Methods In the early stage of the experiment,high-throughput sequencing was performed on five groups of clinical gastric adenocarcinoma tissues and adjacent tissues to screen out the circrNA hsa_circ_0000128 with significantly improved expression for subsequent research,and the downstream miR-623 was predicted by circBank,TargetScan and other databases.Real-time fluorescent quantitative PCR(RT-PCR)was used to detect the expression of hsa_circ_0000128 in 40 clinical gastric adenocarcinoma tissues,adjacent tissues,gastric cancer cell lines(MGC-803,HGC-27,BGC-823)and normal gastric mucosal epithelial cell line GES-1.The two groups with the largest difference in expression were selected for subsequent experiments.Small interfering RNA si_circ_0000128 and si_NCs(transfected with unrelated sequences)were transfected into gastric cancer cell lines,and RT-qPCR was used to verify the interference efficiency.The proliferation ability of each group was determined by clone formation assay.Transwell assay was used to determine the migration ability of gastric cancer cells in each group.Western blotting assay was used to detect the expression levels of apoptosis-related proteins cleaved-caspase-3,Bcl-2 and Bax to verify cell apoptosis.The expression of miR-623 in tissues and cells was verified by luciferase assay.The binding target information was verified by adding miRNA corresponding interference and grouping them.The changes of cell function in each group of gastric cancer cell lines were compared by cell function experiment and Western blotting.PCR and Western blot were used to verify the relationship between the expression level of eukaryotic translation in

关 键 词：hsa_circ_0000128 胃腺癌 miR-623 真核翻译起始因子4A1 细胞凋亡 

分 类 号：R735.2[医药卫生—肿瘤]