YTHDF2通过识别m6A甲基化修饰的LINC00473参与先天性巨结肠的机制研究

Hirschsprung disease (HSCR) is a congenital gut motility disorder that is caused by deficits in migration and proliferation of enteric neural crest cells. We found in a lncRNA chip study that the expression level of LINC00473 was drastically decreased in HSCR patients. Knockdown of LINC00473 significantly affected the proliferation and migration of SK-N-BE(2) cells, indicating that LINC00473 is implicated in the pathogenesis of HSCR. Human YTH domain family 2 (YTHDF2) protein recognizes the N6-Methyladenosine (m6A) modification on RNA and regulates its stability. It is reported that LINC00473 could be recognized and bound with YTHDF2. We found in cell model that YTHDF2 negatively regulated the expression level of LINC00473. Further, we found that the LINC00473 may affect cell migration by competing with miR-195. Low level of LINC00473 may release miR-195 to down regulate DIEXF, which lead to impaired cell migration. This study will thoroughly investigate the role and mechanism of YTHDF2/LINC00473/miR-195/DIEXF in the impaired cell migration and proliferation seen in HSCR.

先天性巨结肠（HSCR）是一种肠神经嵴细胞迁移和增殖障碍导致的先天性消化道畸形。课题组前期在芯片研究中发现LINC00473在病变组织中低表达，细胞实验证明敲低LINC00473可抑制神经细胞的增殖和迁移，提示LINC00473低表达可能与HSCR发生相关。通过生物信息学网站预测提示LINC00473可能发生m6A甲基化，其低表达可能与m6A甲基化识别蛋白YTHDF2介导RNA降解有关。通过RNAi实验发现YTHDF2对LINC00473具有负性调控作用。进一步，课题组通过生物信息学网站预测及细胞实验发现低表达的LINC00473可能作为ceRNA，通过释放miR-195，抑制靶基因DIEXF表达，从而抑制细胞增殖和迁移。本研究将深入研究YTHDF2通过识别并降解m6A甲基化修饰的LINC00473,通过调节miR-195/DIEXF从而参与HSCR发生的机制。

先天性巨结肠（Hirschsprung disease，HSCR）是一种肠神经系统发育异常导致的先天性消化道畸形。LINC00473在HSCR组织中低表达，其可以促进细胞的增殖和迁移。m6A甲基化是RNA修饰的普遍形式，其水平在HSCR中下降。m6A甲基化识别蛋白YTHDF2可降低靶基因的稳定性。RIP实验证实YTHDF2可直接与LINC00473结合，由此提出假设:YTHDF2与LINC00473结合可能通过降低其稳定性下调LINC00473的表达。胞浆胞核分离实验证实LINC00473位于胞浆中，提示其发挥ceRNA作用的可能。低表达的LINC00473可以通过miR-195抑制靶基因DIEXF的表达，从而抑制细胞增殖和迁移。本研究将深入研究YTHDF2通过识别m6A甲基化修饰的LINC00473影响肠神经嵴细胞迁移和增殖在HSCR中的作用机制，为疾病的诊断和治疗提供新思路。