miR-30a靶向调控TET1介导GDNF羟甲基化参与先天性巨结肠发生的分子机制

Aberrant pattern of DNA methylation is observed in intestinal samples of children with Hirschsprung’s disease (HSCR), yet factors involved in this dynamic process remain to be elucidated. In the previous studies, we examined the expression level of TET1 (ten-eleven translocation 1), the regulatory gene coding for hydroxylases, in intestinal samples of HSCR. A significant increase of TET1 expression was found in both expansive and narrow bowel segments of HSCR compared with those in control group, along with an obvious upregulation of GDNF and 5-hydroxymethylcytosine (5hmC, the marker of hydroxymethylation). Considering the previous reports of miR-30a participation in TET1 expression in idiopathic pulmonary fibrosis, we will, based on our previous findings, apply multiple molecular biological technologies in cellular experiments to validate the modulation of miR-30a on the targeted gene TET1, determine the expression levels of miR-30a and targeted genes of GDNF in HSCR intestinal segments. Meanwhile, to investigate the potential influence of GDNF hydroxymethylation on cellular biological behaviors, cell proliferation, cell apoptosis and invasion assay will be documented in the isolated and cultured enteric neural stem cells. Our study aims to verify the important role of miR-30a regulation of TET1 mediated GDNF hydroxymethylation in HSCR, provide novel hints for studying the mechanism of the pathogenesis and progression of HSCR, and highlight theoretical basis for the interference of microRNAs on preventing HSCR.

先天性巨结肠患儿病变肠段存在DNA甲基化修饰异常，然而调控甲基化动态平衡的因素尚未明确。在前期研究中，我们检测了病变肠段DNA羟甲基化关键酶基因TET1的表达，发现患儿扩张肠段和狭窄肠段的TET1显著高于对照组，且狭窄段GDNF表达明显增加及5hmC水平（羟甲基化）上升。结合既往文献报道特发性肺纤维化中miR-30a对于TET1的调控作用，我们拟在前期工作基础上，在细胞水平采用多种分子生物学技术验证miR-30a对于TET1的靶向调控作用，检测先天性巨结肠病变肠段miR-30a及GDNF下游靶基因的表达变化，并在分离培养的肠神经干细胞中检测GDNF羟甲基化对于细胞增殖、细胞凋亡及细胞侵袭等生物学行为的影响，旨在明确miR-30a调控TET1介导的GDNF羟甲基化修饰在先天性巨结肠中的重要作用，为先天性巨结肠发生发展的机制研究提供新的线索，为干预miRNAs以预防该疾病的发生提供理论依据。

先天性巨结肠患儿病变肠段组织存在DNA甲基化修饰异常，然而调控甲基化动态平衡的因素尚未明确。在本课题研究中，我们运用qPCR、免疫组化及Western blot等方法检测了巨结肠患儿狭窄病变段、扩张段和正常对照肠段中DNA羟甲基化酶基因TET1的表达，发现患儿狭窄段和扩张段的TET1显著高于与对照组，且狭窄段GDNF 5hmC水平（羟甲基化）上升，其表达明显增加。结合既往文献报道miR-30a对于TET1的调控作用，我们通过双萤光报告基因系统检测miRNA(hsa-miR-30a-3p)和miRNA(hsa-miR-30a-5p)对TET1的抑制作用，结合定点突变技术确定了microRNA-30a与靶基因TET1 3’UTR 区域的作用位点。采用qPCR检测患儿狭窄段与扩张段miR-30a的表达水平，并与对照组比较，发现HSCR患儿狭窄段和扩张段的miR-30a的表达量均低于正常对照肠段。在采用SD孕大鼠分离培养的肠神经干细胞中，通过慢病毒感染成功构建ENSCs-GDNF（过表达）细胞株后，运用CCK-8细胞增殖检测、软琼脂克隆形成实验、细胞凋亡酶活性检测、流式细胞仪测定细胞周期及Transwell基质胶侵袭实验等方法，发现TET1介导GDNF羟甲基化促进肠神经干细胞的增殖、克隆形成、侵袭等生物学行为。采用Western blot法检测GDNF下游靶基因Akt（p-Akt）、P38（p- P38）及P44/42（p- P44/42）的表达，结果发现AKT和P44/42的磷酸化活性比值在狭窄和扩张段亦比对照组高。本课题研究明确了miRNA-30a调控TET1介导的GDNF羟甲基化修饰在HSCR中的重要作用，为HSCR发生发展的机制研究提供了新的线索。鉴于miRNAs在血液中的稳定表达，我们的研究同时也为干预miRNAs以预防HSCR的发生提供实验基础和理论依据。.