MiR-19b Targeting SOCS3 In Intestinal Epithelial Cell And Is Associated With The Pathogenesis Of Intestinal Mucosal Healing

Objective Inflammatory bowel disease(IBD)is a group of diseases included ulcerative colitis(UC)and Crohn's disease(CD),and the etiology of IBD is not fully understood.Immunologic derangement of intestinal mucosa is believed to contribute to the prevalence,development and outcome of IBD,and intestinal epithelia cells(IECs)are more of importance in regulating the immunological homeostasis of intestine.However,the regulatory mechanisms of homeostasis in IECs remain largely unknown.Recent researches indicate that aberrant micro RNA(miRNA)expression profile has been frequently observed in IBD.Mi RNA could participate in the pathology of IBD by targeting gene expression in a post transcriptional manner.Suppressor of cytokine signaling(SOCS3)could regulate the activation of signal transducer and activator of transcription 3(STAT3)in IEC to influence cell proliferation as well as mucosal healing.It is a pleiotropic cytokine indicating persistent inflammation of intestinal mucosal.Our present research discovered that miR-19 b was less expressed in the inflammatory lesions of CD than that in normal control,while SOCS3 was highly-expressed in CD.Furthermore,SOCS3 was found to be a target of miR-19 b and post-transcriptional controled by miR-19 b in vitro.This study was determined to further explore the role of miR-19 b in intestinal inflammation by TNBS-induced colitis model.Immunohistochemical(IHC),in situ hybridization(ISH)and cell experiments were performed to investigate whether miR-19 b could influence the proliferation of IEC by targeting the expression of SOCS3 and phosphorylation of STAT3.Methods 1.Trinitro-benzene-sulfonic acid(TNBS)was administrated to female BALB/c mouse through anus to induce colitis model which was similar with CD.2.Mi R-19 b was delivered to TNBS-treated mouse.Immunofluorescence was conducted to reveal the location of ectogenic miR-19 b.Then,intestinal inflammation and expression of SOCS3 and miR-19 b were detected and compared.3.Distribution of miR-19 b in intestinal mucosal of CD patiens and normal control was performed by RNA in situ hybridization(ISH).4.Signal transducer and activator of transcription 3(STAT3)and phosphorylated-STAT3(p-STAT3)in intestinal mucosal of objects were determined by immunohistochemical(IHC)in parafin sections.5.Level of sequence complementarity between miRNA and target site of SOCS3 mRNA 3'UTR was validated by luciferase reporter assay.6.The expression of SOCS3 protein and mRNA were detected by western blotting and q RT-PCR after overexpression or knockdown of miRNA in HT-29 cells.7.Protien levels of STAT3 and p-STAT3 were detected by WB in HT-29 cells with upregulated or downregulated of miR-19 b after IL-6 stimulation for 30 minutes.8.The expression of cyclin D1 protein and mRNA were detected by WB and q RT-PCR with overexpression or knockdown of miR-19 b after IL-6 stimulation for 24 h in HT-29 cells to investigate the possible mechanism associated with cell proliferation.9.Flow cytometry and CCK8 were conducted for the detection of cell cycle and proliferation ability according to the instructions with upregulated or downregulated of miR-19 b in HT-29 cells after IL-6 stimulation for 24 h.Results 1.Compared to ethanol control group,TNBS treated mouse showed more severe inflammation in colon.DAI score greately increased in TNBS-treated mouse than that in ethanol group.The colon of TNBS mouse presented hyperemia,edema and ulcer.Histological level by HE staining of colon sections was more significant in macroscopic inflammation.These appearance showed more severe inflammation in TNBS-treated mice than the control,indicating our successful animal models.2.Mouse administrating of miR-19 b in TNBS-treated group showed improvement in symptoms,DAI and HE score.Infused miR-19 b could enter into intestinal epithelium conformed by immunofluorescence.Level of miR-19 b in TNBS-treated mouse was less than ethanol group,while expression of SOCS3 protein was increased in TNBS-treated mouse.The imbalance of miR-19 b and SOCS3 in TNBS-induced colitis was similar with that in CD patiens.Mouse administrating of miR-19 b had elevated miR-19 b while decreased level of SOCS3.3.Using RNA ISH,miR-19 b was hardly detected in IECs in CD patients and obviously in normal control,which was the same as our present research.4.STAT3 was similar expressed in IECs of CD and control by IHC testing,while expression of p-STAT3 was decreased in CD patients.5.The luciferase reporter assay confirmed that miR-19 b directly combined with the 3'UTR of SOCS3 mRNA in HT-29 cells.6.Overexpression of miR-19 b in HT-29 cells could downregulate the protein level of SOCS3,but not that of SOCS3 messenger RNA(mRNA),indicating post-transcriptional control by miR-19 b.7.Downregulation of SOCS3 by miR-19 b mimic caused phosphorylation of STAT3,and upregulating of SOCS3 by miR-19 b inhibitor desreased p-STAT3.Total levels of STAT3 were not significantly different in both groups.8.Expression of cyclin D1 of both protein and mRNA was induced in cells treated with miR-19 b mimic,which was the down target of STAT3 and associated with cell proliferation and cell cycle.On the contrary,downregulation of miR-19 b in HT-29 cells could restrain the level of cyclin D1.9.Cell proliferation and proportion of cells in G1/S transformation was increased in cells with overexpressed miR-19 b.On the contrary,downregulation of miR-19 b showed opposite results.Conclusion Downregulation of miR-19 b and upregulation of SOCS3 were discovered in both CD patiens and TNBS-induced colitis mouse.Administration of miR-19 b in TNBS mouse showed less colon inflammation,indicating the possible pathogenicity of miR-19 b and SOCS3 in CD.Mi R-19 b could targete the expression of SOCS3 in HT-29 cells and influence the levels of p-STAT3 and cyclin D1,to regulate the cell cycle and proliferation.This pathway might act as an regulator of mucosal healing in CD.