miR-126在胃癌中的表达及其靶基因Crk的鉴定

miR-126 expression and its target gene Crk identification in gastric cancer

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 detect the expression of miR-126 in gastric cancer and identify its target genes so as to elucidate the function of miR-126 in the carcinogenesis of gastric cancer. Methods The differential expression of miR- 126 in gastric cancer cell lines, normal gastric mucosa and immortalized normal gastric mucosal epithelial cell line was detected by qRT-PCR, miR-126 expression levels were determined in sixty paired gastric cancer tissues and the relation- ship with clinicopathological variables was analyzed. The target genes of miR-126 were predicted by bioinformatic analysis and validated by luciferase reporter assay, qRT-PCR and Western blot assays were performed to measure the expression level of target genes regulated by miR-126. Results The expression of miR-126 in gastric cancer cell lines was found significantly down-regulated compared with normal gastric mucosa and immortalized normal gastric mucosal epithelial cell line. In addition, the expression level of miR-126 in cancer tissues of 60 patients with gastric cancers was significantly lower than that in its matched non-tumor tissues. Further analysis showed that the gastric cancer tissues with low-expres- sion of miR-126 was associated with larger tumor size, deeper local invasion, more lymph node metastasis, and advanced TNM stage. Bioinformatic analysis suggested that a potential target gene of miR-126 was Crk, in which a putative miR-126 binding site was present within its 3＇UTR. The regulation of Crk gene by miR-126 was further verified by the luciferase reporter assay. It was also demonstrated with qRT-PCR and Western blot analysis that miR-126 could down-regulate Crk expression. Conclusions miR-126 may be as a novel biomarker for gastric cancer, and Crk may be its target gene.