miR-106a-5p靶向调控ERK2对胃癌细胞顺铂耐药性的影响及其分子机制  

Effect of miR-106a-5p on drug resistance of gastric cancer cells to cisplatin through targeted regulation of ERK2

作　　者：杨万荷 李家群 张东艳[1] 王昌高 昝慧 YANG Wanhe;LI Jiaqun;ZHANG Dongyan;WANG Changgao;ZAN Hu(Hainan General Hospital,Haikou 570100,China;不详)

机构地区：[1]海南省人民医院,海口570100 [2]海口市人民医院

出　　处：《山东医药》2021年第17期14-19,共6页Shandong Medical Journal

基　　金：海南省卫生计生行业科研项目(18A201106)。

摘　　要：目的观察微小RNA106a-5p(miR-106a-5p)通过靶向调控细胞外信号调节激酶2(ERK2)对胃癌细胞顺铂(DDP)耐药性的影响,并探讨其可能的分子机制。方法建立胃癌细胞系MGC-803 DDP耐药细胞株MGC-803/DDP,采用RT-PCR法检测MGC-803及MGC-803/DDP中miR-106a-5p及ERK2的表达。在线生物信息学软件star‐Base和Targetscan7.2预测miR-106a-5p与ERK2是否存在结合位点,采用荧光素酶报告实验进一步验证二者的靶向调控关系。将MGC-803/DDP分为mimic组(转染miR-106a-5p mimic)、ERK2组(转染pcDNA3.1-ERK2)、mimic+ERK2组(共转染miR-106a-5p mimic和pcDNA3.1-ERK2)、NC组(不转染),采用EdU染色实验观察各组细胞增殖能力,流式细胞术检测细胞凋亡水平,Transwell实验检测细胞侵袭能力,Westernblotting法检测增殖凋亡相关蛋白(CyclinD1、Caspase3)、上皮间质转化相关蛋白(E-cadherin、N-cadherin、Vimentin)及耐药基因MDR1表达变化,MTT法检测细胞对DDP的耐药性。结果MGC-803/DDP细胞中miR-106a-5p相对表达量低于MGC-803细胞,ERK2 mRNA相对表达量高于MGC-803细胞(P均<0.05)。预测显示,miR-106a-5p与ERK2存在连续结合位点;荧光素酶报告实验结果显示,miR-106a-5p可靶向负调控ERK2。EdU阳性细胞数ERK2组>NC组>mimic+ERK2组>mimic组,细胞凋亡数mimic组>NC组>mimic+ERK2组>ERK2组,贴壁细胞数ERK2组>NC组>mimic+ERK2组>mimic组(P均<0.05)。细胞中CyclinD1、N-cadherin、Vimentin、MDR1表达ERK2组>mimic+ERK2组>NC组>mimic组,Caspase3、E-cadherin表达mimic组>mimic+ERK2组>NC组>ERK2组(P均<0.05)。DDP对细胞的半数抑制浓度ERK2组>NC组>mimic+ERK2组>mimic组(P均<0.05)。结论miR-106a-5p通过靶向抑制ERK2表达降低胃癌细胞对DDP的耐药性,该机制可能与改变增殖凋亡相关蛋白表达、逆转上皮间质转化过程、降低耐药基因MDR1水平有关。Objective To observe the effect of microRNA-106a-5p(miR-106a-5p)on the drug resistance of gastric cancer cells to cisplatin(DDP)through targeted regulation of extracellular signal-regulated kinase 2(ERK2),and to explore its possible molecular mechanism.Methods Gastric cancer cell line MGC-803 DDP-resistant cell line MGC-803/DDP was established.QRT-PCR was used to detect the expression of miR-106a-5p and ERK2 in gastric cancer cell line MGC-803 and drug-resistant cell line MGC-803/DDP.The online bioinformatics software starBase and Targetscan7.2 were used to predict the binding site of miR-106a-5p and ERK2,and luciferase report experiment was used to verify the targeted regulation between them.The drug-resistant cell line MGC-803/DDP was divided into the mimic group(transfected with miR-106a-5p mimic),ERK2 group(transfected with plasmid pcDNA3.1-ERK2),mimic+ERK2 group(co-transfected with miR-106a-5p mimic and pcDNA3.1-ERK2 plasmid),and NC group(blank control group).EdU assay was used to observe the cell proliferation ability of each group.Flow cytometry was used to detect the level of apoptosis in each group.Transwell test was used to detect the cell invasion ability of each group.Western blotting was used to detect the expression changes of proliferation and apoptosis-related proteins Cyclin D1,Caspase-3,epithelial-mesenchymal transition-related proteins E-cadherin,N-cadherin and Vimentin,and multiple drug resistance gene(MDR1).MTT assay was used to detect drug resistance of cells to DDP.Results The relative expression of miR-106a-5p was significantly lower,while ERK-2 mRNA expression level was significantly higher in the MGC-803/DDP cells than those of the MGC-803 cells(both P<0.05).Forecast showed that there was a binding site between ERK2 gene sequence and miR-106a-5p.The results of the luciferase experiment showed that miR-106a-5p could negatively regulate ERK2 expression.EdU positive cell number was in the following order:ERK2 group>NC group>mimic+ERK2 group>mimic group,the number of apoptosis cells:mimic gr

关 键 词：胃癌 miR-106a-5p 细胞外信号调节激酶2 顺铂 化疗耐药 增殖凋亡相关蛋白 上皮间质转化 耐药基因 

分 类 号：R735.2[医药卫生—肿瘤]