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0.276±0.019、0.273±0.018),差异均有统计学意义(t=9.06、7.51,P=0.012、0.017)。双荧光素酶检测结果显示,转染miR-124-3p模拟物的野生型AHR MKN-45细胞中荧光强度低于转染阴性对照模拟物的细胞(0.293±0.020比1.000±0.032),差异有统计学意义(t=18.56,P<0.001)。RT-qPCR检测结果显示,转染miR-124-3p模拟物的MKN-45细胞中AHR和CTNNB1的mRNA水平均低于未处理的MKN-45细胞(0.51±0.09比1.02±0.02、0.46±0.03比1.03±0.01),差异均有统计学意义(t=4.51、16.60,P=0.046、0.004)。蛋白质印迹实验结果显示,转染miR-124-3p模拟物的MKN-45细胞中的AHR和β-联蛋白的蛋白质相对表达量均低于转染0.9%氯化钠溶液和阴性对照模拟物的细胞(3332.94±81.25比9041.60±439.79、8276.54±562.52,2725.79±167.57比9701.94±410.02、8081.66±275.84),差异均有统计学意义(t=15.49、7.91、17.35、19.42,P=0.004、0.016、0.003、0.003)。结论miR-124-3p与胃癌诊断和预后相关,可通过负向调节AHR mRNA和蛋白质表达,进而下调CTNNB1 mRNA和β-联蛋白通路相关蛋白质表达,于体内外诱导胃癌细胞凋亡。因此,miR-124-3p可能成为胃癌诊断和预后评估的潜在标志物。
Objective 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 expression of miR-124-3p was positively correlated with OS,DSS and PFI(R^(2)=1.00,0.99 and 0.99,P=0.029,0.044 and 0.049).The results of subcutaneous tumorigenesis experiment in mice demonstrated that the number of apoptotic cells in the tumor of miR-124-3p group was more than that of negative control group and sodium chloride control group((43.33±1.86)/high power field(HPF)vs.(20.00±1.73)/HPF and(18.67±1.76)/HPF),and the differences were statistically significant(t=8.55 and 8.33,P=0.013 and 0.014).The results of immunohistochemistry showed that the optical density of AHR protein in mice tumor tissue of miR-124-3p group was lower than that of negative control group and sodium chloride control group(0.081±0.008 vs.0.276±0.019 and 0.273±0.018),and the differences were statistically significant(t=9.06 and 7.51,P=0.012 and 0.017).The results of dual luciferase assay indicated that the fluorescence intensity in wild-type AHR MKN-45 cells transfected with miR-124-3p simulant was lower than that of negative control group(0.293±0.020 vs.1.000±0.032),and the difference was statistically significant(t=18.56,P<0.001).The results of RT-qPCR demonstrated that the mRNA levels of AHR and CTNNB1 in MKN-45 cells transfected with miR-124-3p simulant were both lower than those in untreated MKN-45 cells(0.51±0.09 vs.1.02±0.02,0.46±0.03 vs.1.03±0.01),and the differences were statistically significant(t=4.51 and 16.60,P=0.046 and 0.004).The results of Western blotting experiments showed that the relative protein expression levels of AHR andβ-catenin of MKN-45 cells transfected with miR-124-3p simulant were lower than those of transfected with 0.9%sodium chloride solution and negative control simulant(3332.94±81.25 vs.9041.60±439.79 and 8276.54±562.52,2725.79±167.57 vs.9701.94±410.02 and 8081.66±275.84),and the differences were statistically significant(t=15.49,7.91,17.35 and 19.42,P=0.004,0.016,0.003 and 0.003).Conclusions MiR-124-3p is correlated with diagnosis and prognosis of gastric cancer.MiR-124-3p induces apoptosis of gastric cancer cells in vitro and vivo by negatively regulating AHR expression at mRNA and protein level,thereby down-regulating the expression of CTNNB1 mRNA andβ-catenin pathway-related protein.Therefore,miR-124-3p may become a potential diagnostic and prognostic marker of gastric cancer.
作者
王斯炜
张吉翔
董卫国
Wang Siwei;Zhang Jixiang;Dong Weiguo(Department of Gastroenterology,Renmin Hospital of Wuhan University,Wuhan 430061,China)
出处
《中华消化杂志》
CAS
CSCD
北大核心
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)。
