The Mcrornas Profiles In The Colonic Mucosa Of Ibs Patients

Backgroud and Aims:Irritable bowel syndrome(IBS) is a common kind of functional gastrointestinal disease,whose clinical manifestations are characterized by abdominal pain,bloating or abdominal discomfort accompany with altered bowel habits. Based on the excrement character, IBS is divided into three different subtypes,including diarrhea-predominant IBS (IBS-D), constipation-predominant IBS (IBS-C) and mix-IBS (IBS-M). The pathophysiology of IBS is poorly understood.And the diagnosis of IBS is carried out to meet the Rome Ⅲ criteria without standard and objective diagnostic markers. Therefore, searching for convenient and effective biomarkers will be beneficial to diagnosis and treatment of the disease.MicroRNA (miRNA) are endogenous noncoding RNA molecules of18-23nt. Its expression has tissue-cell specific, development temporality and evolution conservation.It can regulate the expression of target genes in a post-transcriptional manner. Evidence indicates that miRNAs play essential roles in pathological and physiological process,inclulding cell proliferation, differentiation,apoptosis,metabolism,oxidative stress, human diseases formation and so on. Recent researches. on IBS have found that abnormal expressions of miRNAs are present in tissues of IBS patients, which suggest miRNAs may play a role in the pathologic process of IBS.Our research aims to screen significantly differentially expressed miRNAs from sigmoid-colon tissues samples between IBS-D and IBS-C microarray platform was used. Then, bioinformatics was applied to predict miRNAs targets and explored the roles of miRNAs played in the disease, laying the foundation for molecular diagnosis and therapies in IBS.Materials and Methods1. Chip were used to screen miRNAs in3sigmoid colon biopsy samples from healthy controls, IBS-D and IBS-C separately.2. Total RNA was extracted from tissue using mirVanaTM RNA Isolation Kit. The concentration of all total RNA samples were measured by agarose gel electrophoresis (AGE).3.Agilent Human MicroRNA Array (8*60K,V19.0) was used to screen miRNA(QuantoBio technology company supplied). Feature Extraction and Gene Spring12.0software were applyed to analyze data,and expression difference>2-fold or<-2.0-fold was used as a threshold to search differentially expressed miRNAs.4.TargetScan was used to predict the target genes of the selected miRNAs.Then the biological function of these target genes was confirmed by GO (gene ontology) overrepresentation and KEGG pathway analyses.5.To validate the microarray data, the expression of eight miRNAs, which were selected based on its biological function,were measured by qRT-PCR using blood samples from an independent set of20patients with IBS-D,10patients with IBS-C and10healthy controls.Results1.It was found that, compared with healthy control, Sixty-Seven miRNAs were significantly up-regulated and five were significantly down-regulated in IBS-D patients, whereas seventeen other miRNAs obviously increased in IBS-C subjects. Then comparion IBS-D with IBS-C,31miRNAs were identified significantly differently expressed, including28miRNAs up-regulated and3miRNAs down-regulated.2.Used TargetScan software to predict targets genes of the selected miRNAs,we found that there were72miRNAs with461target genes in IBS-D/N,17miRNAs with218target genes in IBS-C/N,and31miRNAs with357target genes in IBS-D/IBS-C.Then, GO term analysis showed that the predicted target genes of these miRNAs were significantly involved in ion channel activity (including calcium channel,chloride channel and potassium channel),regulation of cytoskeleton,cell adhesion,across membrane transportion,mast cell granule,neurotransmitter release and signal transduction. KEGG pathway analyses showed that these target genes were enriched in13,7and19pathways respectively (p<0.05), and participated in the regulation of actin cytoskeleton and myosin cytoskeleton, cytokine-cytokine receptor interaction and so on.3. Based on the biological function, eight miRNAs were chosen to enlarge the sample verification by qRT-PCR. It was found that, compared with healthy control,the expression levels of miR-4660,miR-3151,miR-29b-1-5p, miR-4755-3p,miR-3158-3p,miR-4421and miR-3190-3p were significantly up-regulated in the IBS-D group (P<0.05), whereas the expression levels of miR-3151was significantly up-regulated in the IBS-C group (P<0.05). The differential expression of up-regulated (miR-4660, miR-29b-1-5p) miRNAs in ISB-D and IBS-C samples (P<0.05).To sum up,the results demonstrated that these miRNAs expression are in accordance with the microarray.Conclusions1.This study argued that miRNAs are expressed differently in the intestinal mucosa of different subtypes IBS patients’,and the intestinal mucosa of IBS patients’have a specific miRNA expression profiles. And it might suggest that further analyses with miRNAs as a diagnosis and classification marker of IBS are warranted.2.The target genes of differentially expressed miRNAs were determined in IBS-D/N、IBS-C/N and IBS-D/IBS-C.And these target genes are involved in ion channel activity, regulation of cytoskeleton,cell adhesion,across membrane transportion,mast cell granule,neurotransmitter release and signal transduction, which might importantly participate in the IBS pathophysiologic process. Therefore, we consider that miRNAs might play important roles in IBS and their differential expressional pattern is mainly responsible for the diverse clinical manifestation of this disease.