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目的分析microRNA-30a(miR-30a)在胃癌中的表达水平并鉴定其新的靶基因。方法选取29例胃癌患者的肿瘤组织和癌旁组织,利用qRT-PCR检测miR-30a的表达。通过miRNA靶基因预测数据库Target Scan预测miR-30a的靶基因。构建含有miR-30a结合位点的3’UTR片段的荧光素酶载体(p MIR-FAPα),通过荧光素酶报告实验对预测靶基因进行检测。利用Western blot和qRT-PCR对预测靶基因成纤维活化蛋白α(fibroblast activation proteinα,FAPα)的表达进行检测。结果 miR-30a在胃癌组织样本中的表达明显下调,通过生物信息学预测(Target Scan)和荧光素酶实验表明miR-30a mimic可与FAPα基因结合,qRT-PCR和Western blot结果表明miR-30a mimic可抑制FAPαmRNA和蛋白质水平的表达。结论 miR-30a在人胃癌组织样本中表达下调,FAPα是其靶基因。
Objective To analyze the expression of microRNA-30a (miR-30a) in gastric cancer and identify its new target gene. Methods Tumor tissues and adjacent tissues of 29 gastric cancer patients were selected and the expression of miR-30a was detected by qRT-PCR. Target genes of miR-30a were predicted by Target Scan, a miRNA target gene prediction database. The luciferase vector (p MIR-FAPα) containing the 3’UTR fragment of the miR-30a binding site was constructed and the predicted target gene was detected by luciferase reporter assay. Western blot and qRT-PCR were used to detect the expression of target gene fibroblast activation protein α (FAPα). Results The expression of miR-30a in gastric cancer tissues was significantly down-regulated. Target Scan and luciferase assays showed that miR-30a mimic could bind to FAPα gene. QRT-PCR and Western blot showed that miR-30a mimic inhibits the expression of FAPalpha mRNA and protein levels. Conclusion miR-30a is down-regulated in human gastric cancer tissue samples, and FAPα is its target gene.