微小RNA-146b-5p通过Robo1对胆囊癌细胞增殖和凋亡的影响  

The effect of microRNA-146b-5p on the proliferation and apoptosis of gallbladder cancer cells through Robo1

作　　者：丁月超 马超 郑鹏 周闯[2] 黄涛 Ding Yuechao;Ma Chao;Zheng Peng;Zhou Chuang;Huang Tao(Department of Hepatobiliary and Pancreatic Surgery,the Affiliated Cancer Hospital of Zhengzhou University(Henan Cancer Hospital),Zhengzhou 450008,China;Department of Hepatobiliary and Pancreatic Surgery,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)

机构地区：[1]郑州大学附属肿瘤医院(河南省肿瘤医院)肝胆胰外科,郑州450008 [2]郑州大学第一附属医院肝胆胰外科,郑州450052

出　　处：《中华实验外科杂志》2023年第10期2004-2007,共4页Chinese Journal of Experimental Surgery

摘　　要：目的探讨微小RNA(miRNA,miR)-146b-5p通过Robo1对胆囊癌细胞增殖和凋亡的影响。方法选取郑州大学第一附属医院2017年1月到2021年12月手术切除的59例胆囊癌及其癌旁组织作为研究对象,采用荧光定量聚合酶链反应(PCR)分析肿瘤组织和癌旁组织miR-146b-5p表达水平;采用免疫组织化学分析两种组织Robo1蛋白表达水平。采用转染试剂转染miRNA对照和miR-146b-5p抑制剂至人胆囊癌细胞系GBC-SD,分别命名为miRNA对照组和miR-146b-5p KD组,采用噻唑蓝(MTT)和克隆形成实验分析两组细胞活力和增殖能力;采用划痕实验、Transwell和蛋白质印迹法(Western blot)分析两组细胞迁移、侵袭和上皮-间充质转化能力;采用生物信息学和荧光素酶报告基因分析miR-146b-5p的靶基因;采用Western blot分析靶基因表达水平。计量数据比较采用t检验。结果胆囊癌组织miR-146b-5p表达水平(1.01±0.16)明显低于癌旁组织(1.94±0.30),差异有统计学意义(t=20.860,P<0.05)。miRNA对照组细胞吸光度值高于miR-146b-5p KD组(2.22±0.10比1.76±0.06,t=9.294,P<0.05),miRNA对照组细胞克隆形成率高于miR-146b-5p KD组[(85.75±5.16)%比(63.20±5.66)%,t=7.210,P<0.05],miRNA对照组细胞划痕愈合率高于miR-146b-5p KD组[(72.85±5.91)%比(55.56±6.53)%,t=4.806,P<0.05],miRNA对照组细胞迁移数量多于miR-146b-5p KD组[(110.83±16.18)个比(75.66±11.70)个,t=4.313,P<0.05],miRNA对照组细胞E-钙黏蛋白(E-cadherin)蛋白表达水平低于miR-146b-5p KD组(0.94±0.06比1.30±0.17,t=5.081,P<0.05),miRNA对照组细胞N-钙黏蛋白(N-cadherin)和SNAIL蛋白表达水平高于miR-146b-5p KD组(1.28±0.10、0.91±0.09比0.84±0.08、0.52±0.10,t=8.436、7.256,P<0.05),差异均有统计学意义。Robo1是miR-146b-5p的靶基因。miRNA对照组细胞Robo1蛋白表达水平(1.19±0.16)明显高于miR-146b-5p KD组(0.64±0.11),差异有统计学意义(t=6.881,P<0.05)。胆囊癌组织Robo1蛋白表达水平(1.06±0.13)明显高于癌旁组织(0.60±0Objective To investigate the effect of microRNA(miR)-146b-5p on the proliferation and apoptosis of gallbladder cancer cells through Robo1.Methods 59 cases of gallbladder cancer and its adjacent tissues surgically removed from the First Affiliated Hospital of Zhengzhou University from January 2017 to December 2021were selected as the research subjects.The expression levels of miR-146b-5p in tumor and adjacent tissues was analyzed by fluorescence quantitative polymerase chain reaction(PCR).The expression levels of Robo1 protein in tumor and adjacent tissues was analyzed by Western blotting.Using transfection reagents to transfect miRNA control and miR-146b-5p inhibitors into the human gallbladder cancer cell line GBC-SD,they were named miRNA control group and miR-146b-5p KD group,respectively.The cell viability and proliferation ability of the two groups were analyzed using methyl thiazolyl tetrazolium(MTT)and clone formation experiments.The migration,invasion,and epithelial mesenchymal transformation abilities of the two groups of cells were analyzed by wounding heal,transwell and Western blotting.Analyze the target genes of miR-146b-5p using bioinformatics and dual luciferase reporter genes.The expression level of target genes was analyzed by Western blotting.The comparison of measurement data adopts t-test.Results The expression level of miR-146b-5p in gallbladder cancer tissue(1.01±0.16)was significantly lower than that in adjacent tissues of gallbladder cancer(1.94±0.30,t=20.860,P<0.05).As compared with cells in the miR-146b-5p KD group,The light absorption value of cells in the miRNA control group was significantly higher(2.22±0.10 vs.1.76±0.06,t=9.294,P<0.05),the clone formation rate was significantly higher[(72.85±5.91)%vs.(55.56±6.53)%,t=4.806,P<0.05],the scratch healing rate was significantly higher[(72.85±5.91)%vs.(55.56±6.53)%,t=4.806,P<0.05],the migration number of cells was significantly higher[(110.83±16.18)cells vs.(75.66±11.70)cells,t=4.313,P<0.05],the expression level of E-cadherin prote

关 键 词：微小RNA-146b-5p ROBO1 胆囊癌 增殖 凋亡 

分 类 号：R735.8[医药卫生—肿瘤]