MiR-9 Targeting SNIP1 In Intestinal Epithelial Cell And Is Associated With The Pathogenesis Of Crohn's Disease

Background&AimCrohn's disease(CD)is one of the major types of IBD and until now we have little knowledge about its etiology.Environmental,immune,genetic and intestinal micro flora factors are closely related with occurrence and development of CD.In response to the stimuli of pathogenic factors,intestinal epithelial cells(IECs)elicit rapid changes of gene expression patterns,which induce the damage of gut mucosal barrier and thereby lead to the pathogenesis of CD.Therefore,maintenance of epithelial homeostasis has a pivotal role in improving clinical outcomes of CD.Current researches indicate that distinct microRNA(miRNA)profile has been observed in CD,and the aberrantly expressed miRNAs could participate in the pathology of IBD by targeting gene expression and regulating the homeostasis of the intestinal mucosal barrier.SMAD nuclear interacting protein 1(SNIP1)regulates stability of Cyclin D1 mRNA.Previous research has shown that SNIP1 expression was decreased in the intestinal mucosal tissue of IBD patients,therefore SNIP1 may be associated with IBD.However,the research of how SNIP1 contributes to CD has just beginning and the precise mechanism is not clear.Our previous study found that miR-9 was significantly upregulated in intestinal tissues of CD.The present study was determined to further investigate whether miR-9 could disturb epithelial homeostasis via targeting SNIP 1 expression and subsequently triggering Cyclin D1 degradation in CD by clinical sample analysis,cell experiments and experimental colitis model.Methods:1.Inflammation-related miRNAs levels were assayed in intestinal tissues from active CD patients and healthy controls by real-time quantitative reverse transcriptase-polymerase chain reaction(qRT-PCR)to detect differentially expressed miRNA.And we chose miR-9 to have further investigation.2.Targetscan and miRanda software predicted that SNIP1 is the potential gene target of miR-9.Dual immunofluorescence staining and qRT-PCR were used to examine the expression of the target gene SNIP1 in CD and healthy control tissues.3.Level of sequence complementarity between miR-9 and target site of SNIP1 mRNA 3,UTR was validated by luciferase reporter assay.4.The protein and mRNA levels of SNIP1 were detected by Western Blotting(WB)and qRT-PCR after overexpression or knockdown of miR-9 in HT-29 cells.The mRNA of CCND1,which is the downstream gene of SNIP1,was also examined by qRT-PCR.Cyclin D1,CCDN1 encoding protein,was examined by WB.5.The expression of miR-9 and SNIP1 was detected in TNF-a treated HT-29 cells.6.CCK-8 assay and flow cytometry assay were applied to detect cell proliferation ability and cell cycle after upregulating or downregulating miR-9 in HT-29 cells.7.Female BALB/c mice aged 6-8 weeks were administrated 2,4,6-trinitrobenzene-sulphonic acid(TNBS)to induce colitis which mimicking CD.To validate the anti-inflammatory role of miR-9 inhibitor,firstly mice were treated with miR-9 inhibitor.Then the intestinal tissues samples were assessed with macroscopy,disease activity index(DAI)values and hematoxylin-eosin(HE)staining histopathological study.8.The expressions of miR-9 and SNIP1 mRNA were also examined in TNBS-induced colitis mouse..Results:1.Among inflammation-related miRNAs,miR-9 level was significantly increased in intestinal tissue of CD patients compared to normal controls.2.Bioinformatics prediction showed that SNIP1 was the target gene of miR-9.qRT-PCR confirmed that SNIP1 mRNA were downregulated in CD.And dual immunofluorescence staining demonstrated the downregulated expression of SNIP1 in IECs of CD.3.The luciferase reporter assay confirmed that miR-9 directly combined with the 3'UTR region of SNIP1.4.In HT-29 cells,over-expression of miR-9 resulted in the mRNA level of SNIP1 and CCND1 as well as the protein level of SNIP1 and Cyclin D1 decreased,vice versa.These results indicate that a post-transcriptional regulation exists.5.The expression of miR-9 was increased in TNF-? treated HT29 cells and SNIP1 level was decreased which were time-varying.6.Cell proliferation and proportion of cells in G1/S transformation were decreased in cells with overexpressed miR-9.On the contrary,downregulation of miR-9 showed opposite results.7.Compared to ethanol control group,TNBS treated mice showed more severe inflammation in colon,increased DAI score and higher histological level by HE staining,indicating that TNBS successfully induced colitis.Mice administrating of miR-9 inhibitor in TNBS-treated group showed improvement in macroscopy,DAI and HE score,indicating that miR-9 inhibitor had an inverse correlation with murine colitis.8.Level of miR-9 in TNBS-treated group was higher than ethanol group,while expression of SNIP1 mRNA was decreased in TNBS-treated group.The imbalance of miR-9 and SNIP1 in TNBS-induced colitis was similar with that in CD patients.Mice administrating of miR-9 inhibitor had decreased miR-9 level while increased mRNA level of SNIP 1.Conclusion:Downregulation of miR-9 and upregulation of SNIP1 were examined in both CD patients and TNBS-induced colitis mouse,which indicating the possible pathogenicity of miR-9 and SNIP1 in CD.MiR-9 could target the expression of SNIP1 in HT-29 cells and influence the levels of cyclinDl,to regulate the cell cycle and proliferation.This pathway might act as an important regulator of mucosal healing in CD.