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目的探讨循环型micro RNA-92a(mi R-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(q RT-PCR)检测血清mi R-92a m RNA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mi R-92a,噻唑蓝法检测细胞的增殖活性,q RT-PCR、Western blot检测CDX2基因的表达。结果 HD患儿血清中mi R-92a m RNA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mi R-92a后,CDX2 m RNA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 mi R-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。
Objective To investigate the expression of circulating miRNA-92a (mi R-92a) in Hirschsprung ’s disease (HD) and to inhibit the proliferation of enteric neural stem cells (ENSCs) by upregulating the transcription factor CDX2 Possible mechanism. Methods Preoperative serum samples from 16 patients with HD who underwent radical hirschsprung radical surgery at Yishui Central Hospital of Shandong Province from January 2013 to December 2015 were selected. Real-time fluorescent quantitative polymerase chain reaction (q RT-PCR) was used to detect the expression of mi R-92a m RNA in serum. ENSCs were extracted from fetal intestine of 15-day-old pregnant rats, and ENSCs were identified by immunological method. Nestin + ENSCs were sorted by immunomagnetic beads method. MiR-92a was overexpressed in Nestin + ENSCs by lipofection. Activity, q RT-PCR, Western blot detection of CDX2 gene expression. Results The relative expression of mi R-92a m RNA in serum of HD patients was (23.5 ± 4.66), which was higher than that of the control group (t = 12.661, P = 0.000). Nestin staining was positive in early stage of ENSCs culture, (13.024 ± 3.882), which was higher than that of control group (F = 47.212, P = 0.000), CDX2 (P = 0.000), and the expression of CDX2 mRNA in ENSCs overexpressing mi R- The protein expression was (0.436 ± 0.0828) higher than the control group (F = 48.793, P = 0.000). The proliferative activity of the cells was significantly inhibited at 24, 48 and 72 h after transfection. Conclusion mi R-92a may promote the development and progression of HD by up-regulating the expression of CDX2 and inhibiting the proliferation of ENSCs.