微RNA-124-3p通过芳香烃受体激活β-联蛋白通路调控胃癌细胞增殖和侵袭的机制  

The mechanism of microRNA-124-3p in regulating proliferation and invasion of gastric cancer cell by aryl hydrocarbon receptor-activated β-catenin pathway

作　　者：王斯炜 张吉翔[1] 董卫国[1] Wang Siwei;Zhang Jixiang;Dong Weiguo(Department of Gastroenterology,Renmin Hospital of Wuhan University,Wuhan 430061,China)

机构地区：[1]武汉大学人民医院消化内科,武汉430061

出　　处：《中华消化杂志》2022年第9期619-626,共8页Chinese Journal of Digestion

基　　金：国家自然科学基金 (82170549)。

摘　　要：目的探讨微RNA(miR)-124-3p及其靶向基因芳香烃受体(AHR)在胃癌诊断和预后评估中的价值,及其调控胃癌细胞增殖和侵袭的相关分子机制。方法基于癌症基因组图谱数据库和基因型组织表达数据库获得miR-124-3p在胃腺癌患者的临床和预后特征,通过生物信息学分析miR-124-3p水平高低与不同病理分期、TNM分期、总生存期、无病生存期和无进展间期胃腺癌患者的关系。由Target Scan 7.1网络工具预测miR-124-3p与互作分子AHR mRNA的作用位点,通过小鼠皮下成瘤实验、免疫组织化学染色、双荧光素酶检测、实时定量聚合酶链反应(RT-qPCR)、蛋白质印迹实验验证miR-124-3p在AHR mRNA中的靶结合位点。选择9只4~6周龄、体重为(18.43±0.29)g的雄性Balb/c裸小鼠,分别由小鼠尾静脉注射miR-124-3p模拟物(miR-124-3p组)、阴性对照模拟物(阴性对照组)和0.9%氯化钠溶液(氯化钠对照组),用miR-124-3p模拟物、阴性对照模拟物和0.9%氯化钠溶液转染胃癌细胞(MKN-45、AGS),分析生物学实验中3组小鼠AHR和连环蛋白β1基因(CTNNB1)的mRNA表达水平,AHR和β-联蛋白的蛋白质表达水平,以及miR-124-3p对转染的胃癌细胞增殖和侵袭的影响。统计学方法采用Pearson相关分析和Holm-Sidak法校正的多重t检验。结果miR-124-3p低表达与胃腺癌患者严重的病理分期、TNM分期均呈正相关(R^(2)=0.83、0.86,P=0.031、0.023);miR-124-3p高表达与总生存期、无病生存期和无进展间期延长均呈正相关(R^(2)=1.00、0.99、0.99,P=0.029、0.044、0.049)。小鼠皮下成瘤实验结果显示,miR-124-3p组瘤体内的凋亡细胞数多于阴性对照组和氯化钠对照组[(43.33±1.86)/高倍视野比(20.00±1.73)/高倍视野、(18.67±1.76)/高倍视野],差异均有统计学意义(t=8.55、8.33,P=0.013、0.014)。免疫组织化学染色结果显示,miR-124-3p组小鼠体内肿瘤组织的AHR蛋白质的光密度低于阴性对照组和氯化钠对照组(0.081±0.008比0Objective To explore the value of microRNA(miR)-124-3p and its target gene aryl hydrocarbon receptor(AHR)in the diagnosis and prognostic evaluation of gastric cancer,and the related molecular mechanisms in regulating proliferation and invasion of gastric cancer cell.Methods The clinical and prognostic characteristics of patients with gastric adenocarcinoma expressing miR-124-3p were obtained from The Cancer Genome Atlas database and Genotype-Tissue Expression database.The correlation between miR-124-3p expression level and pathological stage,TNM stage,overall survival(OS),disease-specific survival(DSS)and progression-free interval(PFI)in patients with gastric adenocarcinoma were studied by bioinformatics analysis.The interaction sites between miR-124-3p and AHR mRNA were predicted by Target Scan 7.1 online tool.The target binding sites of miR-124-3p in AHR mRNA were verified by subcutaneous tumorigenesis experiment in mice,immunohistochemistry,dual luciferase assay,quantitative real time-polymerase chain reaction(RT-qPCR)and Western blotting.Nine male Balb/c nude mice,aged 4 to 6 weeks with weight of(18.43±0.29)g were injected with miR-124-3p simulant(miR-124-3p group),negative control simulant(negative control group)and 0.9%sodium chloride solution(sodium chloride control group)through the tail vein.Gastric cancer cell lines(MKN-45,AGS)were transfected with RNA simulants(including miR-124-3p simulant,negative control simulant and 0.9%sodium chloride solution).The expression of AHR and Cateninβ1 gene(CTNNB1)at mRNA level,the expression of AHR andβ-catenin at protein level in 3 mice groups and the effects of miR-124-3p transfection on the proliferation and invasion of transfected gastric cancer cells were analyzed.Pearson correlation analysis and Holm-Sidak corrected multiple t test were used for statistical analysis.Results Low expression of miR-124-3p was positively correlated with severe pathological stages and TNM stages in patients with gastric adenocarcinoma(R^(2)=0.83 and 0.86,P=0.031 and 0.023).High exp

关 键 词：微RNA-124-3p 胃肿瘤 受体 芳基烃 生物标记 细胞凋亡 

分 类 号：R735.2[医药卫生—肿瘤]