1、MicroRNA MiR-19b Targeting SOCS3 In Colonic Epithelial Cells And Is Associated With The Pathogenesis Of Crohn’s Disease 2、The Expression And Significance Of Th17 Cells And Th17-relatedCytokines In Active UC Patients

Background:Crohn’s disease (CD) is a chronic recurrent inflammatory disorder of the gastrointestinal tract with unknown etiology. Its incidence may be related to immune, genetic, environment, microbial, mucosal barrier, internal environment and other factors. The abnormal immune response plays a critical role in the development of the disease, and become the focus of recent research. As a member of the SOCS family, supreesor of cytokine signaling-3(SOCS3) involved in inflammation and immune responses through negative regulation of cytokine signaling, and is associated with a variety of inflammatory and immune-related diseases. Currently, many studies have showed that increased SOCS3 expression has been detected in intestinal epithelial cells and a variety of cells in lamina propria of CD patients, and may be related to the pathogenesis of CD.MicroRNAs (miRNAs) are a class of noncoding single strand molecule RNAs, consisting of 20-24 nucleotides in length. They play an important role in regulating gene expression at post-transcriptional level by base-pairing to the 3’ untranslated region (3’UTR) of target mRNAs, which leads to block the translation or trigger the degradation of the target mRNAs. Then they can participate in a variety of basic biological processes, such as ontogeny, apoptosis, proliferation and differentiation, and they are closely linked to the development of a variety of diseases. Numerous studies show that there are specific miRNAs expression profiles in serum and tissue of CD patients. The aim of our study was to investigate the contribution of SOCS3 expression-associated miRNA to the regulation of chemokines production in colonic epithelial cells(CEC) in active CD.Aim:1. To screen miRNAs which can target SOCS3 in colonic mucosa of active Crohn’s disease(CD).2. To observe the production of IL-6-induced chemokines affected by the expression of SOCS3 in CEC which can be regulated by the miRNAMethods:1. Targetscan Human 6.2 was used to screen miRNAs which can target the 3’ untranslated region (3’UTR) of SOCS3. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was used to detect these miRNAs and SOCS3 mRNA levels, and western blotting was used to detect SOCS3 protein levels in active CD and healthy subjects.2. The luciferase reporter assay was performed to confirm the target gene.3. The expressions of SOCS3 mRNA and protein were determined by quantitative PCR and western blotting after over-expression or down-regulation miRNA in Caco2 cells.4. Caco2 cells were stimulated with recombinant human IL-6(rhIL-6) for 24h and 72h after over-expression or down-regulation miRNA and SOCS3. The levels of chemokines were measured by enzyme linked immunosorbent assay(ELISA).Results:1. In the eight miRNAs screened by Targetscan, only miRNA-19b expression was decreased, while SOCS3 protein expression was increased in affected area of colonic mucosa tissue in active CD. There was an inverse correlation between the expression of miR-19b and SOCS3 protein. Bioinformatic prediction showed that SOCS3 was the target gene of miR-19b.2. The luciferase reporter assay confirmed that miR-19b directly recognized 3’UTR of SOCS3.3. In vitro, knockdown of miR-19b increased SOCS3 expression, and over-expression of miR-19b decreased SOCS3 expression.But SOCS3 mRNA levels can not be changed.4. Down-regulation of miR-19b decreased MIP-3a production induced by IL-6 modulated by SOCS3.Conclusion:MiR-19b plays a critical role in regulating production of chemokines by targeting SOCS3 in colonic epithelial cells.Background:Ulcerative Colitis (UC) is a chronic non-specific inflammatory disorders of the colon. Its pathogenesis may be associated with immune, genetic, microbiological, environmental, and immune in the development of the disease is essential. Recent studies have found that Th17 cells and Th17-related cytokines interleukin-17 (IL-17), interleukin-21 (IL-21) highly expressed in the colon of UC patients. And they can induce inflammatory response and damage to the intestinal mucosa through regulation of adaptive and innate immune system to participate in the development of UC.Aim:To investigate the association of distribution of Th17 cells and the expressions of Th17-related cytokines(IL-17 and IL-21) with disease activity, CRP, ESR, endoscopic stage, and histological grading in patients with active ulcerative colitis (UC)Methods:40 patients with active UC and 20 healthy controls were recruited. The infiltration of Th17 cells in colonic mucosa were observed by immunofluorescence staining. The situ expressions of IL-17 and IL-21 were evaluated by a standard immunohistochemical procedure. The serum levels of IL-17 and L-21 were determined by ELISA.Results:1. Compared with healthy controls, the number of Th17 cells was mainly increased in the lamina propria of active UC patients.2. The expressions of IL-17 and IL-21 in the colonic mucosa and serum in active UC patients were significantly higher than control (P<0.05).The expressions of IL-17 and IL-21 were increasing from mild to severe UC.3. In active UC patients, IL-17 and IL-21 were positively correlated with CRP(P<0.05). And the expression of IL-17 was also positively correlated with ESR significantly (P<0.05).4. In active UC patients, IL-17 and IL-21 were positively correlated with endoscopic stage and histological grading, respectively (P<0.05).Conclusion:Th17 cells and Th17-related cytokines (IL-17 and IL-21) were increased in the colonic mucosa in active UC patients, Thl7 cells and Thl7-related cytokines may play an important role in disease activity and mucosal damage.