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目的:分析miR-126在胃癌中的表达水平并鉴定miR-126的靶基因,以阐明miR-126在胃癌发生机制中的功能。方法:采用qRT-PCR分别检测miR-126在胃癌细胞株、正常胃黏膜组织、永生化胃黏膜上皮细胞、胃癌及配对癌旁组织的表达水平,并与胃癌组织的临床病理指标进行相关性分析。采用生物信息学方法预测出miR-126的靶基因,并通过荧光素酶报告系统加以验证;采用qRT-PCR及Western印迹法检测miR-126对靶基因mRNA及蛋白质表达水平的影响。结果:qRT-PCR检测结果显示,miR-126在胃癌细胞株中的表达水平明显低于其在正常胃黏膜组织及永生化胃黏膜上皮细胞株的表达水平。miR-126在60例病人的胃癌组织中表达的水平显著低于其在配对癌旁组织中表达的水平;且胃癌组织miR-126表达水平低者,肿瘤组织体积较大,胃壁浸润较深,易发生淋巴结转移,病理分期也较晚。生物信息学分析提示Crk mRNA的3′UTR含有miR-126直接作用的靶序列,荧光素酶报告系统检测进一步验证了该靶序列,qRT-PCR及Western印迹法证实miR-126对Crk蛋白表达的调控发生在转录后水平。结论:miR-126有望成为研究胃癌的新型标志物;miR-126通过对其靶基因Crk的调控参与了胃癌的发生、发展过程。
OBJECTIVE: To analyze the expression level of miR-126 in gastric cancer and to identify the target gene of miR-126 in order to clarify the function of miR-126 in the pathogenesis of gastric cancer. Methods: The expression of miR-126 was detected by qRT-PCR in gastric cancer cell lines, normal gastric mucosa, immortalized gastric mucosa epithelial cells, gastric cancer and paracancerous tissues, respectively. The correlations were analyzed with the clinicopathological parameters of gastric cancer . The target genes of miR-126 were predicted by bioinformatics methods and verified by luciferase reporter system. The effect of miR-126 on mRNA and protein expression of target genes was detected by qRT-PCR and Western blotting. Results: The results of qRT-PCR showed that the expression level of miR-126 in gastric cancer cell lines was significantly lower than that in normal gastric mucosa and immortalized gastric mucosa epithelial cell lines. The expression level of miR-126 in gastric cancer tissue of 60 patients was significantly lower than that in paired paracancer tissues. The expression of miR-126 in gastric cancer tissue was low, the volume of tumor tissue was larger, the gastric wall infiltration was deeper, Lymphatic metastasis, pathological staging is also late. Bioinformatics analysis suggested that the 3’UTR of Crk mRNA contained a direct target sequence of miR-126. The luciferase reporter system verified the target sequence further. QRT-PCR and Western blot confirmed the expression of Crk protein Regulation occurs at post-transcriptional level. Conclusion: miR-126 is expected to become a new marker of gastric cancer. MiR-126 participates in the carcinogenesis and progression of gastric cancer by regulating its target gene Crk.