MicroRNAs Expression Profiling Analysis In Hirschsprung Disease By Microarray

BackgroundHirschsprung disease (HSCR), which was also called congenital megacolon or intestinal aganglionosis, was a congenital anomaly of the large intestine arising from a developmental abnormality of the enteric nervous system (ENS). The pathology of HSCR was characterized by the absence of ganglion cells attributed to a failure of migration of neural crest cells in the myenteric and submucosal plexuses of the distal intestine.HSCR was one of the most common congenital digestive malformation and had an incidence of around 1:5000 live births, ranking second in the digestive tract malformations. And its incidence was higher in Asian populations. Currently, the pathogenesis of HSCR was consistent with the view that an early stage embryo microenvironmental changes and genetic factors both played an important role in hirschsprung disease, so the common role of environmental factors and genetic factors leaded to the occurrence of HSCR. There were currently many genes known to be associated with HSCR, such as RET、GDNF、NRTN、ECE1、EDN3、SOX10、 PH0X2B、NRG1,and so on.In recent years, the genetic study of HSCR had made significant progress, researcher for the first time reported case of the total colonic HSCR children associated with interstitial deletion of the long arm of chromosome 10 in 1992, and further confirmed that it may be located between 10q11.2 and 10q21.2. Finally, other study confirmed that the existence of RET gene mutations in children with HSCR in 1994. RET proto-oncogene, known as tyrosine kinase receptor, was widely expressed in neural crest cell, which was crucial for the development of enteric neurons, and functional experiment had confirmed that RET gene knockout mice could result in the absence of ganglion cells of entire digestive wall. So, RET might play an important role in the pathogenesis of HSCR. GDNF gene complementary to RET gene had also been found mutations in HSCR patients. The expression of EDNRB accompanied the whole process of embryonic development, and its function was to make the neural crest cells develop into mature ganglion cells, and animal experiments had found that targeted destruction of the EDNRB gene could lead to the absence of ganglion cells ganglion cells of the bowel. It had been confirmed that its corresponding ligand EDN3 mutations might play the important role in the process of HSCR. Swenon et al reported the EDN3 genes in 66 cases of sporadic and 9 cases of familial HSCR cases, discovering a new heterozygous mutations in exon 2, this framework mobile mutations could lead to codon stop early, as a result, it would lead to a non-functional gene product. SOX10 also proved to be HSCR susceptibility genes, it was expressed in embryonic period from neural crest cells, involved in the formation of the peripheral-nervous system, and many researchers had identified SOX gene mutation in a large number of children with HSCR. The study showed that the gene plays an important role in the process of differentiation and the migration of neural crest stem cells, the genetic defect can significantly interfere with migration and differentiation of neural crest cells. As was known to all, ENCCs proliferated rapidly while migrating toward and within the gut. Moreover, a subpopulation of ENCCs undergoed neuronal differentiation as they migrated within the gut wall to form a plexus. The colonization of the gut by ENCCs required coordinated proliferation, migration, and neuronal differentiation, as perturbations to cell number, migratory behavior, or rate of neuronal differentiation could result in aganglionosis in animal models.MicroRNAs (miRNAs) were single-stranded, non-coding RNA molecules that were about 21 to 25 nucleotides long, they were endogenous small molecule RNA. MiRNAs post-transcriptionally regulated gene expression by binding to the 3’ untranslated region (3’UTR) of their target messenger RNAs (mRNAs) through base-pairing, which in turn triggered mRNA degradation or translational inhibition.To date, thounds of miRNAs had been identified and many of them had been implicated in numerous biological processes, such as cell proliferation, migration, organ development, stress reaction and apoptosis and miRNAs were reported to have strong association with diverse diseases. MiRNA was critical to the regulation of the development and maintenance of healthy neurons, expression of many miRNAs was dynamically regulated during neurogenesis, neuronal maturation and brain development. In mice, deletion of Dicer resulted in deficits in brain development, failure to develop appropriate neuronal phenotype, neuronal atrophy, severe growth defects and early death. The ENS was the largest part of the peripheral nervous system, and there were at least as many neurons in the gut as there were in the spinal cord. So, miRNA might be closely related to the development of the enteric nervous system, and some study had reported that some miRNAs might be involved in the process of cell differentiation, proliferation, migration and apoptosis of the ENCCs. And in the process of development, the synergy of the positive and negative gene transcription and the transcription regulation created by miRNA leaded to cell diversity, and once this synergy obstacles, congenital dysplasia might be produced, leading to the occurrence of congenital diseases.Part I Screening the miRNA expression profiles in the bowels of patients with. HSCR using microarrayObjective:To investigate the differentially expressed miRNAs and preliminarily establish the differential miRNAs expression profiling between spastic segments and distending segments in the patients with HSCR using microarray.Methods:27 cases of fresh HSCR-stenosed segments and HSCR-dilated segments colon tissue samples from patients with HSCR, who underwent surgical treatment in Zhujiang Hospital, were collected.Total RNA was isolated using TRIzol (Invitrogen) and purified with RNeasy mini kit (QIAGEN) according to manufacturer’s instructions in 6 HSCR specimens, including spastic segments and distending segments. Then RNA quality and quantity was measured by using nanodrop spectrophotometer (ND-1000, Nanodrop Technologies) and RNA Integrity was determined by gel electrophoresis. After quality control, the miRCURYTM Hy3TM/Hy5TM Power labeling kit (Exiqon,) was used according to the manufacturer’s guideline for miRNA labelling, the Hy3TM-labeled samples were hybridized on the miRCURYTM LNA Array (v.18.0) (Exiqon) according to array manual after stopping-the labeling procedure. Scanned images were then imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction. Replicated miRNAs were averaged and miRNAs that intensities>=30 in all samples were chosen for calculating normalization factor. Expressed data were normalized using the Median normalization. After normalization, significant differentially expressed miRNAs between two groups were identified through Fold change and P-value. Differentially expressed miRNAs between two samples were filtered through Fold change.Results:The result of total RNA quality identification showed that the total RNA A260/ A280 ratios were close to 2.0, and A260/A230 ratios were greater than 1.8, suggesting that the segments were high-quality without degradation or contamination of other impurities, such as DNA, proteins, etc. and they were suitable for microarray experiments. Significantly differentially expressed miRNAs between HSCR-stenosed segments and HSCR-dilated segments colon tissue samples were obtained using microarray. Compared to ganglionic segment tissues,19 miRNAs(miR-3941, miR-K12-1-3p, miR-K12-3-3p, miR-145-3p and so on) were significantly up-regulated more than 2 fold,7 miRNAs(miR-625-5p, miR-520g-3p, miR-1260a, miR-3916 and so on) were significantly down-regulated more than 2 folds in aganglionic segment tissues (both P<0.05).Conclusion:There were differentially expressed miRNAs between HSCR-stenosed segments and HSCR-dilated segments colon tissue samples. These significantly differentially expressed miRNAs might play an important role in the pathogenesis of HSCR, which might serve as a novel approach for further revealing the pathogenesis of HSCR in the future.Part II Predicting the target genes of miRNA using target gene prediction programsObjective:To predict the target genes of miRNA by using target gene prediction programs, and to preliminarily explore the mechanism of miRNAs contributed to the pathogenesis of the patients with HSCR.Methods:On the basis of Fold Change value, P value and ForeGround value (ideal value> 100), we predicted the target genes of eight significantly differentially expressed miRNAs. We used Medical Subject Headings and keywords (including HSCR, Hirschsprung disease, congenital megacolon or intestinal aganglionosis) as search terms in PubMed website, focusing on genes related to HSCR. Then we analysised the relationship between these eight significantly differentially expressed miRNAs and genes related to HSCR Using Targetscan, miRTarBase, miRDB, MicroRNA.org target gene prediction software.Results:We found the target genes of these 8 significantly differentially expressed miRNAs using target gene prediction programs, and these genes had been shown to be associated with the pathogenesis of HSCR. The bioinformaties study illustrated that there were 1 target gene for miR-193a-3p,5 target genes for miR-3646,4 target genes for miR-1260a,1 target gene for miR-4524b-5p,3 target genes for miR-145-3p, 3 target genes for miR-4505.Conclusion:6 miRNAs were complementary to genes associated with HSCR, showing these miRNAs might be involved in the pathogenesis of HSCR by binding to these target genes.Part III Validation of differentially expressed miRNAs in the segment tissues of patients with HSCR by using quantitative real-time PCRObjective:To further validate differentially expressed miRNAs which were obtained from Microarray chip experiments and complementary to genes associated with HSCR between HSCR-stenosed segments and HSCR-dilated segments colon tissue samples using quantitative real-time polymerase chain reaction (qRT-PCR).Methods:Total RNAs, including miRNAs, were extracted from 27 HSCR-stenosed segments (HSCR-S) and HSCR-dilated segments (HSCR-D) colon tissue samples, deriving from patients with HSCR who underwent surgical treatment in Zhujiang Hospital. Then we validated the differentially expressed miRNAs using poly(T) adaptor qRT-PCR. We used SYBR (?) Green I chimeric fluorescence to detect the expression of miR-145-3 p, miR-4505 and miR-1260a. Micrornas primers were made up of miRNA-specific primers and Oligo (dT)-Universal Tag universal reverse transcription primer. RNA reverse transcription and real-time PCR reactions were performed according to the instructions. (1)2ug total RNA containing the targeted miRNA was collected and the production of poly(A) tailed in miRNA 3* end were made by using miRcute miRNA First-Strand cDNA Synthesis kit. The reverse transcription reaction was performed by using Oligo (dT)-Universal Tag universal reverse transcription primer,leading to the synthesis First-strand cDNAs, the reaction conditions for this process was 37 ℃,60 min,95 ℃,5 min, the products were immediately frozen and stored at-20 ℃. (2) We added 2μl solution containing miRNA first strand cDNA into miRcute miRNA qPCR Detection kit (SYBR Green) for fluorescence quantitative detection of miRNA. PCR reaction conditions as folows: 94 ℃ 2min,1 cycle,94 ℃ 20s,60 ℃ 34s,45 cycles.Results:QRT-PCR showed the relative expression levels of miR-145-3p (1.42±0.42, aganglionic segment vs.0.90±0.31, ganglionic segment) and miR-4505 (1.30±0.30, aganglionic segment vs.0.76±0.22, ganglionic segment) displayed a statistical difference between groups (both P＜0.001).To find other factors that might affect the expression levels of miRNAs in the HSCR-dilated segments colon tissue, the expression of miRNA in age, HSCR type and gender levels were analysed. The results indicated that the expression levels of miR-145-3p in children over 1 year of age and children under 1 year of age displayed no statistical difference (P=0.570), so did miR-4505 (P=0.034); and that the expression levels of miR-145-3p expressed much more in long-segment HSCR than short-segment HSCR, displaying a statistical difference (P=0.034), so did miR-4505 (P=0.000);and that the expression levels of miR-145-3p in different gender displayed no statistical difference (P=0.464),so did miR-4505 (P=0.766)Conclusions:1. The results obtained from microarray were accordant with those from qRT-PCR, suggesting that abnormal expression of miRNAs was found between HSCR-stenosed segments and HSCR-dilated segments, and miR-145-3p and miR-4505 might be involved in the pathogenesis of HSCR.2. The expression of miR-145-3p and miR-4505 had nothing to do with gender and age levels of children with HSCR, but might be associated with the length of aganglionosis in HSCR. Discussion the role of miR-145-3p and miR-4505 in the process of HSCR would provide new ideas to further explore the function and role of miRNAs in the pathogenesis of HSCR.