摘要
目的研究先天性巨结肠(HSCR)肠管组织和血清微小核糖核酸(miRNA)的表达差异。方法取52例经手术和病理确诊的HSCR患儿无神经节细胞肠管组织和正常结肠组织标本,以及52例HSCR患儿和52例健康婴儿的血清标本(20例用于样本检测,32例肝扩大样本量验证)。应用microRNA Taq Man Low Density Array技术,分别检测无神经节细胞肠管和正常对照结肠肠管、HSCR患儿和健康婴儿血清标本的miRNA表达谱,对肠管组织和血清miRNAs表达谱进行整合分析,确定HSCR无神经节细胞肠管和血清miRNAs特异性表达情况。对肠管组织和血清一致性异常表达的miRNAs首先进行芯片样本独立验证,然后对32对肠管组织和血清样本进行扩大样本量验证,并对一致性异常表达的miRNAs进行靶基因预测。结果与正常结肠组织相比,有47个miRNAs在HSCR无神经节细胞肠管组织中呈差异表达,其中17个miRNAs表达上调,30个miRNAs表达下调;HSCR患儿血清中有32个miRNAs呈差异表达,均为表达上调。miR-218—1和miR-885-5p为在HSCR无神经节细胞肠管和血清中呈一致性差异表达的miRNAs。芯片样本独立验证及扩大样本量验证显示,miR-218-1和miR-885-5p在HSCR无神经节细胞肠管和血清中均呈显著性高表达,差异有统计学意义(miR-218—1:组织芯片样本0.01758±0.00229比0.00337±0.00050,P〈0.001;组织扩大样本量0.01353±0.00174比0.00443±0.00060,P〈0.001。miR-885-5p:组织芯片样本0.00030±0.00011比0.00004±0.000008,P=0.0276;组织扩大样本量0.00459±0.00016比0.00004±0.00001,P=0.0145。miR-218-1:血清芯片样本0.76960±0.28550比0.04514±0.01507,P:0.0155;血清扩大样本量1.15100±0.43000比0.02307±0.00381,P=0.0087。miR-885-5p:血清芯片样本1.59500±0.44170比0.16940±0.03446,P=0.0012;血清扩大样本量1.68900±0.45300比0.14610±0.03124,P=0.0012。生物信息软件预测显示miR-218-1和miR-885-5p的共同靶基因是酪氨酸激酶受体基因RET、PLAGl和NeuroDl。结论HSCR无神经节细胞肠管和患儿血清均存在显著差异表达的miRNAs,其中miR-218-1和miR-885-5p呈一致性高表达,可能与HSCR的发生有关。
Objective To investigate the different expressions of pathological tissue and serum microRNAs (miRNAs) in Hirschsprung disease(HSCR). Methods Pathological colon tissues and serum samples were obtained from 52 confirmed HSCR cases respectively by surgery and pathology and from 52 matched controls, respectively. An initial screening of the tissues and serum microRNA expression were performed through TaqMan Low Density Array. The candidate tissue and serum miRNAs were validated by quantitative real - time - PCR in the 20 paired array samples and extra 32 paired samples after the integration of the screening result. The bioinformatical software online including miR- base, Target Scan, PicTar and MiRanda were used to predict the target mRNA of the consistent microRNAs in the tis- sues and the serum. Results Compared with the controls,g7 microRNAs were differently expressed in HSCR tissues, including 17 up- regulated miRNAs and 30 down -regulated miRNAs;32 upregulated miRNAs were also detected to be differently expressed in the HSCR serum. Among these microRNAs, miR- 218 -1 and miR- 885 -5p were identi- fied to have a consistent significant different expression in both tissues and the serum, which were validated as high - expressed in microarray samples and expanded 32 paired samples( miR -218 - 1 :tissue array 0. 017 58 ± 0. 002 29 vs 0. 003 37 ±0. 000 50, P 〈 0. 001 ; tissue expanded expression 0. 013 53 ± 0. 001 74 vs O. 004 43 ± 0. 000 60, P 〈 0. 001. miR -885 -5p:tissue array 0.000 30 ±0. 000 11 vs 0.000 04 ±0. 0000 08,P= 0.027 6;tissue expanded ex- pression 0.004 59 ±0.000 16 vs 0.000 04 ±0.000 01 ,P =0.014 5. miR -218 - 1 :serum array 0.769 60 ±0.285 50 vs O. 045 14 ±0. 015 07 ,P =0.015 5 ;serum expanded expression 1. 151 00 ±0.430 00 vs O. 023 07 ±0.003 81 ,P = 0. 008 7. miR - 885 - 5p : serum array 1. 595 00 ± 0.441 70 vs O. 169 40 ± 0. 034 46, P = 0.001 2 ; serum expanded expres- sion 1. 689 00 ± 0.453 00 vs 0. 146 10 ± 0. 031 24, P = 0.001 2). Specifically, the target genes of these 2 microRNAs were RET,PIAG1 and NeuroD1 ,which had been reported to be directly related to HSCR. Conclusions Significantly dif-ferential expressed miRNAs exist in the pathological tissue and the serum of HSCR. MiR -218 - 1 and miR -885 -5p, which showing consistent differential expression, may be involved in the pathogenesis of HSCR.
出处
《中华实用儿科临床杂志》
CSCD
北大核心
2016年第5期380-383,共4页
Chinese Journal of Applied Clinical Pediatrics
基金
国家自然科学基金(81370473、81400574、81570467)