lncRNA-XIST调控miR-519/TAB3轴介导结直肠癌EMT和侵袭转移的机制研究

Epithelial-mesenchymal transition (EMT) is key to colorectal cancer (CRC) metastasis. Our previous study have confirmed that miR-519 inhibits the progression of CRC, but its regulatory mechanism is unclear. The previous studies have showed that lncRNA-XIST play an important role in the occurrence and development of tumor. Our preliminary studies suggest that XIST was significantly elevated in the CRC tissues, and the expression of XIST was negatively correlated with miR-519 expression in CRC tissues. XIST knockdown could inhibit EMT, migration and invasion of the CRC cells. In addition, bioinformatics analysis revealed that XIST has the binding sites of miR-519, and TAB3 may be the downstream target gene of miR-519. Furthermore, knockdown of XIST could reduce the expression of TAB3 in CRC cells. Therefore, we speculated that XIST promotes the EMT and metastasis of CRC mediator of by acting as a ceRNA (competing endogenous RNA) for miR-519, thereby increasing expression of its target TAB3. To test this hypothesis and investigate the mechanism of its action, we will construct stale stable transfection cell lines and in vivo tumor model, observe the effect of XIST on EMT and metastasis in CRC. Furthermore, we will clarify the mechanism through which XIST regulates miR-519/TAB3 axis by bioinformatics analysis and molecular biological methods. These results will provide the new theoretical basis for clarifying the mechanism of CRC invasion and metastasis.

上皮间质转化(EMT)是结直肠癌等恶性肿瘤转移的关键所在。我们研究发现miR-519可抑制结直肠癌的发生发展，但其调控机制尚未阐明。研究表明lncRNA-XIST可促进多种恶性肿瘤的侵袭转移。预实验结果显示：XIST在结直肠癌组织中高表达，且与miR-519的表达呈负相关；XIST存在miR-519的结合位点，且下调XIST可抑制结肠癌细胞中miR-519潜在靶基因TAB3的表达；下调 XIST可抑制结直肠癌细胞发生EMT，并降低细胞的侵袭转移能力。据此我们推测：XIST通过竞争性结合miR-519上调TAB3表达进而促进结直肠癌EMT和侵袭转移。本项目通过构建稳定转染细胞株及裸鼠肿瘤在体模型，明确XIST对结直肠癌EMT和侵袭转移的影响。结合生物信息学分析，并通过分子生物学技术阐明XIST调节miR-519/TAB3轴的分子机制。本研究结果将为阐明结直肠癌的侵袭转移机制提供新的理论依据.

上皮间质转化(EMT)是结直肠癌等恶性肿瘤转移的关键所在。本研究旨在阐明lncRNA-XIST调控miR-519/TAB3轴介导结直肠癌EMT和侵袭转移的机制研究。本研究使用细胞分子生物学的方法在高转移能力的LOVO和SW1116结肠癌细胞中敲减XIST或TAB3，癌细胞的转移能力减弱；在低转移能力的HCT116和SW480肠癌细胞中过表达TAB3，癌细胞的转移能力增强。并且敲减XIST能降低TAB3的表达。由于XIST属于长链非编码RNA，而非编码小RNA miR519在XIST有潜在结合位点，并且TAB3的mRNA也有miR519的潜在结合位点，为了验证XIST通过miR519调控TAB3的表达，我们在肠癌细胞中转入miR519的类似物或抑制剂，实验发现miR519类似物降低TAB3的表达，miR519抑制剂促进TAB3的表达，而在敲减XIST的情况下，miR519抑制TAB3的能力更强。RNA免疫沉淀实验表明miR519与TAB3 mRNA及XIST在同一复合物中，进一步说明XIST，miR519和TAB3 mRNA的调控关系。Transwell体外转移实验表明，miR-519抑制肠癌细胞转移， miR-519抑制剂促进转移；在肠癌细胞中敲减XIST增强miR-519抑制剂的作用；而在肠癌细胞中敲减TAB3消除miR-519和miR-519 抑制剂的作用。以上结果说明XIST通过抑制miR519的功能进而促进TAB3的表达，然后促进肠癌细胞转移。裸鼠体内实验证明XIST促进肠癌细胞转移，而miR-519抑制肠癌细胞转移。进一步实验证明XIST通过增强NFkB信号通路促进肠癌细胞转移，而miR-519通过阻止NFkB信号通路抑制肠癌细胞转移，这与TAB3激活NFkB信号通路的结论相符。本研究为进一步了解肠癌转移的分子机理奠定基础。