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

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_circ_0000128的相对表达量明显低于SI-NC组(P<0.05)。SI-circ+miR-inh组miR-623含量较SI-circ+miR-NC组下降(P<0.05)。SI-circ组Cleaved-caspase-3、Bax相对表达量较SI-NC组增加(P<0.05),Bcl-2相对表达量下降(P<0.05)。HGC-27和MGC-803细胞中Si-circ组的克隆形成率[(11.57±1.724)%、(12.47±2.401)%]明显低于Si-NC组[(32.53±3.250)%、(25.07±1.557)%],差异有统计学意义(P<0.05)。加入miR-623 inhibitor后可逆转抑制现象,SI-circ+miR-NC组克隆形成率[(10.43±1.305)%、(12.50±1.400)%]明显低于Si-circ+miR-inh组[(25.03±1.665)%、(19.63±1.955)%],差异具有统计学意义(P<0.01)。Transwell实验表明,SI-circ组较SI-NC组迁移明显降低(P<0.05)。EIF4A1在40对胃癌组织及胃癌细胞株HGC-27、MGC-803表达较癌旁组织、正常胃黏膜上皮细胞GES-1中呈高表达,差异具有统计学意义(P<0.01)。蛋白免疫印迹实验显示当敲减hsa_circ_0000128时,EIF4A1的含量下降;当加入miR-623 inhibitor逆转敲减circRNA情况时,EIF4A1的表达含量上升(P<0.01)。结论hsa_circ_0000128在胃癌细胞株、组织中呈高表达,hsa_circ_0000128作为miR-623的海绵上调EIF4A1促进胃癌细胞的生物学功能;含量下调后可显著抑制胃癌细胞增殖、迁移并诱导其凋亡。

Objective 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 initiation factor 4A1(EIF4A1)and the content of miR-623 in each group.Results The expression of hsa_circ_0000128 in gastric cancer tissues and cell lines was higher than that in adjacent tissues and normal gastric epithelial cells.Knockdown of hsa_circ_0000128 inhibited the proliferation,migration and apoptosis of gastric cancer cells HGC-27 and MGC-803,and decreased the expression of EIF4A1 protein.Conclusion hsa_circ_0000128 is highly expressed in gastric cancer cell lines and tissues.hsa_circ_0000128 acts as a sponge of miR-623 to up-regulate EIF4A1 and promote the biological function of gastric cancer cells.Down-regulation can significantly inhibit the proliferation,migration and induce apoptosis of gastric cancer cells.