循环型microRNA-92a在先天性巨结肠中的表达及上调CDX2基因的研究

Expression of serum microRNA-92a in Hirschsprung's disease and its role in up-regulation of CDX2

摘要:

目的  探讨循环型microRNA-92a（miR-92a）在先天性巨结肠（HD）中的表达，以及通过上调尾侧型同源转录因子-2（CDX2）基因抑制肠神经干细胞（ENSCs）增殖的可能机制。方法  选取2013年1月-2015年12月在山东省临沂市沂水中心医院行先天性巨结肠根治术16例HD患儿的术前血清标本。实时荧光定量聚合酶链反应（qRT-PCR）检测血清miR-92a mRNA的表达。提取孕15 d胎鼠肠管ENSCs，通过免疫学方法鉴定ENSCs；免疫磁珠法分选Nestin+ENSCs，脂质体转染使Nestin+ENSCs内过表达miR-92a，噻唑蓝法检测细胞的增殖活性，qRT-PCR、Western blot检测CDX2基因的表达。结果  HD患儿血清中miR-92a mRNA的相对表达量为（23.5±4.66），高于对照组（t =12.661，P =0.000）；ENSCs细胞培养早期Nestin染色阳性，具有向Tuj-1阳性、胶质纤维酸性蛋白阳性细胞分化的潜能；ENSCs过表达miR-92a后，CDX2 mRNA的相对表达为（13.024±3.882），高于对照组（F =47.212，P =0.000），CDX2蛋白表达为（0.436±0.0828），高于对照组（F =48.793，P =0.000），细胞的增殖活性在转染后24、48和72 h明显受抑制。结论  miR-92a通过上调CDX2基因的表达，抑制ENSCs的增殖，可能促进HD发生、发展。

Abstract:

Objective To investigate the expression of serum miR-92a in Hirschsprung's disease (HD) and its role in regulation of CDX2 and inhibition of enteric neural stem cells (ENSCs). Methods Serum miR-92a was detected by real-time PCR in 16 patients with Hirschsprung's disease from January 2013 to December 2015 in Yishui Central Hospital of Linyi. ENSCs were collected from SD rats of gestational day 15, and identified by immunological method. ENSCs were over-expressed with miR-92a using lipofectin transfection after being separated by magnetic activated cell sorting, and then the expression of CDX2 was valued by real-time PCR and Western blot. The cell proliferation activity was determined by MTT method. Results The level of serum miR-92a mRNA in the patients with Hirschsprung's disease was higher than that in the control group (t = 12.661, P = 0.000). Nestin was positively stained in the ENSCs of early culture stage, which also had the potential to differentiate into double positive cells with Tuj-1 and glial fibrillary acidic protein (GFAP). The over-expression of miR-92a in the ENSCs increased the level of CDX2 mRNA (F = 47.212, P = 0.000) and protein (F = 48.793, P = 0.000) compared to the control group. Cell proliferation activity was inhi-
bited at 24, 48 and 72 h after transfection. Conclusions miR-92a may promote the occurrence and development of HD through up-regulation of CDX2 and consequent inhibition of ENSC proliferation.