Identification And Functional Analysis Of Expression Profiling Of Serum MicroRNA On Patients With Irritable Bowel Syndrome

Objectives:Irritable bowel syndrome(IBS) is a common disease of intestinal disorder. Itsetiology and pathogenesis has not yet been completely clarified.It is lack of specificalmorphological changes and biochemical abnormalities. In the study of microarraytechnology,we analysised and compared serum miRNA expression in IBS and normalcontrol to search for serum miRNA expression profiling of IBS. We used real-timequantitative PCR and bioinformatics analysis to achieve the purpose of identificationand functional analysis on serum miRNA expression profiling of IBS and to lay amolecular basis for the further study of miRNA regulating mechanisms and moleculardiagnostic tools of IBS.Methods:(1) Detection by microarray:We detected pooled serum specimens ofconstipation-predominant IBS(C-IBS), diarrhea-predominant IBS(D-IBS) and normalcontrol by microarray to search for differentially expressing miRNAs.(2) Bioinformatic analysis of miRNA: Based on differentially expressingmiRNAs,we predicted the all target genes which were regulated by differentialmiRNAs in TargetScan6.0database. We did GO annotation and Pathway annotationbased on the NovelBio database to get all GO and Pathway Term which genes wereinvolved in. The Fisher test calculated for significant level of each GO and PathwayTerm to filter out significant GO and Pathway Term in which differential genesenriched. Extract the corresponding genes from concerning significant GO andsignificant Pathway to construct miRNA-target gene network respectively with theircorresponding miRNAs.(3) Analysis by real-time quantitative PCR:We got miRNAs which played coreregulatory role in IBS by bioinformatics analysis.We used quantitative real-time PCRfor analysis of these miRNAs in serum specimens, and the final results of PCR werecompared with these of miRNA microarray. Results:(1) Serum miRNA expression profiling:We screened differentially expressingmiRNAs from the results of microarray by Limma algorithm.Compared with thenormal control,14serum miRNAs of significant differences expressed in patientswith C-IBS,24serum miRNAs of significant differences expressed in patients with D-IBS.Patients with C-IBS and D-IBS expressed of hsa-miR-449a, hsa-miR-210, hsa-miRPlus-I181b-2*(table3.2).(2) Bioinformatic analysis of miRNA: By the bioinformatic analysis, weobtained3groups of significant GO associated with IBS(C-IBS related-nervechanging GO,signal pathway GO, biochemical GO),(D-IBS related-nerve changingGO,signal pathway GO, biochemical GO),(C-IBSand D-IBS common related-neurodevelopment and pain GO, cell motility GO, biochemical GO)(appendix A).Extract the corresponding genes from concerning significant GO and significantPathway to construct miRNA-gene target-network respectively with their correspond-ding miRNAs(figure3.3). We got miRNAs which played core regulatory role innetwork(table3.3) and key target genes regulated by miRNAs.(3) Validation of miRNA by real-time quantitative PCR: We analysised miRNAswhich played a core role in the miRNA-target gene-network by real-time quantitativePCR analysis(figure3.4and table3.4, figure3.5and table3.5, figure3.6and table3.6).The results of real-time quantitative PCR displayed that miRNA expressiontrends were consistent with these of the microarray analysis (figure3.7, figure3.8,figure3.9).Conclusion:(1) We found serum miRNA expression profiling closely related to IBS by themicroarray and bioinformatics analysis.In the C-IBS group more significantlyupregulated miRNAs are hsa-miR-3130-3p, hsa-miR-30c, hsa-miR-555, moresignificantly downregulated miRNAs are hsa-miR-210, hsa-miR-499a, hsa-miR-4289;in the D-IBS group more significantly upregulated miRNAs are hsa-miR-484, hsa-miR-3170, hsa-miR-335, more significantly downregulated miRNAs are hsa-miR-188-5p, hsa-miR-196a, hsa-miR-500a; co-expressed miRNAs of two groups are hsa-miR-210, hsa-miR-499a. (2) MiRNA expression validated by quantitative PCR is consistent with theresults of microarray, which proves that the results of microarray are reliable.(3) Specific serum miRNA expression profiling may provide a new non-invasivebiomarker for diagnosis of IBS.(4) MiRNAs related to patients with IBS may regulate brain-gut axis changes,visceral sensitivity, secretion of intestinal and power of intestinal smooth musclethrough multiple target genes and signaling pathways related to the nervous system,biochemical and cell movement to participate in the pathogenesis of IBS.