Tang Weibing, Qin Jingjing, Tang Junwei, Zhang Hongwei, Zhou Zhigang, Li Bo, Geng Qiming, Wu Wei, Xia Yankai, Xu Xiaoqun
State Key Laboratory of Reproductive Medicine, Institute of Toxicology, School of Public Health, Nanjing Medical University, Nanjing, China.
Cell Physiol Biochem. 2013;32(6):1655-67. doi: 10.1159/000356601. Epub 2013 Dec 5.
MiR-141 has been confirmed to be associated with various human diseases. However, whether miR-141 is involved in the pathogenesis of Hirschsprung's disease (HSCR) remains unknown. Here, we design the experiment to reveal the relationship between miR-141 and HSCR.
Quantitative real-time PCR and Western blot were used to detect the expression levels of miR-141 and its potential genes in 70 tissues of HSCR compared with 60 controls. Bisulfite sequencing PCR (BSP) assay was applied to explain the possible mechanism of the aberrant expression level of miR-141. We employed a dual-luciferase reporter assay to validate the regulation relation between miR-141 and CD47/CUL3. Cell migration, proliferation, apoptosis, and cell cycle progression were examined by transwell assay, MTT assay, and flow cytometry, respectively.
MiR-141 was down-regulated whereas CD47 and CUL3 expression was increased in colon tissues from patients with HSCR compared with control group, The increased level of CD47 and CUL3 induced by miR-141 reduced proliferation and migration of 293T and SH-SY5Y cells. Furthermore, this suppression was reversed by reducing of CD47 and CUL3. Hypermethylation of a CpG Island in the promoter region of miR-141 gene was confirmed in HSCR tissues.
Aberrant reduction of miR-141 may play an important role in the pathogenesis of HSCR with the inhibiting affection on cell migration and proliferation abilities. The present study demonstrates for the first time the role of miR-141 and its target genes in the occurrence of HSCR, and provides us a new direction for the study of the pathogenesis of Hirschsprung's disease.
已证实miR-141与多种人类疾病相关。然而，miR-141是否参与先天性巨结肠病（HSCR）的发病机制仍不清楚。在此，我们设计实验以揭示miR-141与HSCR之间的关系。
与60个对照相比，采用定量实时PCR和蛋白质免疫印迹法检测70个HSCR组织中miR-141及其潜在基因的表达水平。应用亚硫酸氢盐测序PCR（BSP）分析来解释miR-141异常表达水平的可能机制。我们采用双荧光素酶报告基因检测法来验证miR-141与CD47/CUL3之间的调控关系。分别通过Transwell实验、MTT实验和流式细胞术检测细胞迁移、增殖、凋亡及细胞周期进程。
与对照组相比，HSCR患者结肠组织中miR-141表达下调，而CD47和CUL3表达增加。miR-诱导的CD47和CUL3水平升高降低了293T和SH-SY5Y细胞的增殖和迁移能力。此外，降低CD47和CUL3可逆转这种抑制作用。在HSCR组织中证实了miR-141基因启动子区域一个CpG岛的高甲基化。
miR-141的异常降低可能在HSCR的发病机制中起重要作用，对细胞迁移和增殖能力有抑制作用。本研究首次证明了miR-141及其靶基因在HSCR发生中的作用，并为先天性巨结肠病发病机制的研究提供了新方向。
