MiR-192-5p通过调控ERCC3和ERCC4表达逆转胃癌细胞顺铂耐药性的体内研究

发布时间：2018-04-11 04:13

本文选题：miR-192 + 胃肿瘤　；参考：《安徽医科大学》2017年硕士论文

【摘要】：目的为了探索miR-192-5p在体内能否通过调控核苷酸切除修复(NER)途径中ERCC3和ERCC4蛋白表达逆转胃癌细胞的顺铂耐药性。方法采用基因芯片微阵列方法检测胃癌顺铂耐药细胞SGC7901/DDP和亲本细胞SGC7901中LncRNA、mRNA和miRNA的表达差异。通过生物信息学预测软件分析核酸切除修复蛋白ERCC3和ERCC4的mRNA中存在miR-192-5p的靶向结合位点。利用慢病毒在SGC7901/DDP细胞中稳定上调miR-192-5p的表达、在SGC7901细胞中稳定下调miR-192-5p的表达,应用qRT-PCR验证转染后miR-192-5p的表达水平。免疫蛋白印记技术(Western blot)方法检测上调和下调miR-192-5p表达后细胞内ERCC3和ERCC4的表达。用四甲基偶氮唑盐微量酶反应比色法(MTT)检测调控miR-192-5p表达后细胞的顺铂耐药性。将稳定表达miR-192-5p的胃癌细胞、空载对照细胞和未转细胞构建人胃癌裸鼠皮下移植瘤模型,腹腔灌注顺铂,测量各组瘤体的生长曲线及瘤体重量,免疫组化检测ERCC3和ERCC4在瘤体内的表达。结果基因芯片检测结果提示,SGC7901/DDP和SGC7901细胞中存在多个LncRNA、mRNA和miRNA的表达差异。通过生物学软件分析这些异常表达的基因可能与顺铂耐药相关,其中miR-192-5p在SGC7901/DDP中的表达较SGC7901中明显降低,ERCC3和ERCC4的mRNA中存在miR-192-5p的靶向结合位点。上调miR-192-5p后,细胞内ERCC3和ERCC4的表达下调,顺铂的敏感性增强;下调miR-192-5p后,细胞内ERCC3和ERCC4的表达上调,顺铂的耐药性增强。动物模型实验表明,上调miR-192-5p后,瘤体内ERCC3和ERCC4的表达下调,瘤体对顺铂化疗的敏感性提高,而下调miR-192-5p表达后,瘤体内ERCC3和ERCC4的表达上调,瘤体对顺铂耐药。结论MiR-192-5p通过调控NER途径中ERCC3和ERCC4表达影响胃癌细胞的顺铂耐药性
[Abstract]:Objective to investigate whether miR-192-5p can reverse cisplatin resistance in gastric cancer cells by regulating the expression of ERCC3 and ERCC4 protein in the nucleotide excision repair pathway.Methods cDNA microarray was used to detect the expression of LncRNA-mRNA and miRNA in cisplatin resistant gastric cancer cell line SGC7901/DDP and parental cell SGC7901.Bioinformatics prediction software was used to analyze the existence of targeted miR-192-5p binding sites in ERCC3 and ERCC4 mRNA.Lentivirus was used to steadily up-regulate the expression of miR-192-5p in SGC7901/DDP cells and down-regulate the expression of miR-192-5p in SGC7901 cells. QRT-PCR was used to verify the expression level of miR-192-5p after transfection.The expression of ERCC3 and ERCC4 in the cells after up-regulation and down-regulation of miR-192-5p expression was detected by Western blot.The cisplatin resistance of cells regulated by miR-192-5p was detected by microenzyme reaction of tetramethylazolium.Gastric cancer cells expressing miR-192-5p stably, unloaded control cells and untransformed cells were used to construct subcutaneous transplanted tumor model of human gastric cancer in nude mice. Cisplatin was infused intraperitoneally to measure the growth curve and weight of tumor in each group.The expression of ERCC3 and ERCC4 in the tumor was detected by immunohistochemistry.Results the results of gene chip analysis showed that there were differences in mRNA and miRNA expression of LncRNAs in SGC7901 / DDP and SGC7901 cells.The abnormal expression of these genes may be related to cisplatin resistance by biological software. The expression of miR-192-5p in SGC7901/DDP is significantly lower than that in mRNA of SGC7901 and ERCC3 and ERCC4 have targeted miR-192-5p binding sites.After upregulation of miR-192-5p, the expression of ERCC3 and ERCC4 was down-regulated, and the sensitivity of cisplatin was enhanced. After down-regulation of miR-192-5p, the expression of ERCC3 and ERCC4 was up-regulated, and the resistance of cisplatin was enhanced.After up-regulation of miR-192-5p, the expression of ERCC3 and ERCC4 was down-regulated, and the sensitivity of tumor to cisplatin chemotherapy was increased. After down-regulation of miR-192-5p, the expression of ERCC3 and ERCC4 was up-regulated, and the tumor was resistant to cisplatin.Conclusion MiR-192-5p affects cisplatin resistance of gastric cancer cells by regulating the expression of ERCC3 and ERCC4 in NER pathway.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

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