miR-145下调与结直肠癌上皮间叶转化的分子机制探讨

Molecular mechanism by Down-regulation of miR-145 promotes colorectal epithelial mesenchymal transformation

目的:研究miR-145通过靶向调节目的基因对结直肠癌(CRCs)上皮间叶转化(EMT)的影响。方法:通过实时荧光定量PCR检测不同进展CRC肿瘤患者组织样本,验证miRNA差异表达;利用划痕实验检测多株CRC细胞株过表达miR-145后细胞的转移能力;通过Western blot检测过表达miR-145后细胞EMT相关分子标志表达变化;结合生物信息学、Luciferase报告系统以及Western blot预测并验证miR-145的靶基因。结果:miR-145在高转移预后差的CRC肿瘤组织中表达明显上调,且过表达miR-145后CRC细胞株转移能力明显降低,间叶细胞多种分子标志显著下调,而上皮细胞多种分子标志显著上调,其转移能力明显降低。生物信息学预测microRNA靶基因以及实验证实miR-145可以直接靶向调节TGFBR2蛋白表达,是TFG-β信号通路的主要受体及信号分子。结论:miR-145直接靶向调节TGFBR2影响TFG-β通路,抑制EMT过程,进而抑制肿瘤的侵袭和转移。过表达miR-145能够抑制CRC细胞的转移。miR-145是临床诊断CRC的新靶标,对CRC的防治及预后分析具有潜在的临床意义。

Objective-To identify the impact of miR-145 on epithelial mesenchymal transfor- mation（EMT） of colorectal cancer（CRCs） by targeting Regulated target genes. Methods： we veri- fied miRNA differentially expressed by Real-time PCR detecting different progress of CRC tumor tissue samples. We Observed the metastasis of several CRC cell lines after miR-145 overexpressed by scratch assay. The expression change of cell EMT-related molecular markers was detected by Western btot after miR-145 overexpressed. What is more, miR-145 target gene was predicted and validated with bioinformatics, luciferase reporting system as well as Western blot. Results： The expression of miR-145 significantly upregulated in CRC tumor tissue, which had high metastasis and poor prognosis. The metastasis of CRC cell line significantly reduced after miR-145 overexpressed. a variety of molecular markers of mesenchymal cells was significantly decreased, while the epithelial cells multiple molecular mark was significantly increased, and its metastasis significantly reduced. Bioinformatics predicted that microRNA target genes and experiments confirmed that miR-145 can be directly targeting regulatory TGFBR2 protein expression, miR-145 is the major receptor and sig- naling molecule of TFG-β signaling pathway as well. Conclusion： miR-145 impacts TFG-β pathway by target regulating TGFBR2 directly, then inhibits EMT process, finally the invasion and metastasis of tumor are inhibited. The overexpression of miR-145 can inhibit the metastasis of CRC cell. miR-145 is a new target for clinical diagnosing CRC, and has potential clinical significance for preventing and treating CRC.