| Inflammatory bowel disease(IBD)includes ulcerative colitis(UC)and Crohn’s disease(CD).The pathogenesis of IBD has not been fully clerified..Gene,environment,immune responds and intestinal infection may be involved in the pathogenesis of IBD.Intestinal mucosal immune dysfunction,increased secretion of inflammatory cytokines,intestinal flora disorder and intestinal mucosal barrier function destruction are participated in UC.The nuclear factor-κB(NF-κB)signaling pathway regulates the expression of proinflammatory cytokines,and the increased expression of NF-κB related factors in UC patients promotes the expression of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and IL-8.Previous studies show that patients with UC have intestinal epithelial dysfunction,but the specific molecular mechanism is still limited.micro RNA(miRNA)is a kind of single-stranded small molecule non-coding RNA,which is bound to the 3’-untranslated region(3’ UTR)of target gene m RNA by means of base complementary pairing,and participates in the regulation of cell differentiation,maturation and apoptosis by regulating post-transcriptional gene expression.Previous researches have reported that miR-142-5p was upregulated in the mucosal and peripheral circulation of UC patients and played an important role in UC.Suppressor of cytokine signaling 1(SOCS1),an important member of the SOCS family,is a negative regulator that inhibits cytokine signal transduction through feedback.Previous studies have found that SOCS1 plays an important role in a variety of processes such as cell differentiation,inflammation,immune response.SOCS1 is involved in the pathogenesis of UC through the Janus kinase(JAK)/signal transducer and activator of transcription(STAT)signaling pathway and NF-κB signaling pathway.Previous studies on differentiation of macrophages,innate lymphocytes and the pathogenesis of multiple sclerosis have confirmed that SOCS1 is the target gene of miR-142-5p,and it is participated in the cell differentiation process and disease pathogenesis.The purpose of this study is to observe the expressions of miR-142-5p and SOCS1 in patients with UC,analyze the correlation between miR-142-5p and SOCS1 and the effect of miR-142-5p on the inflammatory response of intestinal epithelial cells as an in vitro cell culture model of UC,so as to clarify the molecular regulatory mechanism of UC,provide a new theoretical basis for the treatment of UC.The experiment consists of three parts as follows:Part one The expression of miR-142-5p and SOCS1 in colon tissues of patients with ulcerative colitis.Objective:To detect the expression and interrelation of miR-142-5p and SOCS1 in colon tissues of patients with UC,and evaluate the correlation between them.Methods:The Colonic mucosa tissue samples were collected from 25 patients with active UC(AUC group),19 patients with paracmastic UC(PUC group)and 21 healthy people(NC group).miRNA microarray analysis was carried out according to the standard procedures of Illumina.Relative expression of miR-142-5p was measured by qRT-PCR,and analyzed the relationship among clinical characteristics of UC patients.qRT-PCR and Western Blot were used to examine the expression of the SOCS1 m RNA and protein.The relationship between miR-142-5p and SOCS1 was evaluated by Pearson correlation.Results:miRNA microarray showed that expression of miR-142-5p marked changed in AUC tissues.The relative expression miR-142-5p was significantly up-regulated in AUC group(2.45±0.09)compared with PUC group(1.27±0.07)and NC group(1.01±0.07),P<0.05.The expression of miR-142-5p was associated with severity of UC patients.SOCS1 was decreased in AUC group(0.49±0.20)compared with NC group(0.93±0.18)and PUC group(0.80±0.16),P<0.05.The expression levels of miR-142-5p and SOCS1 revealed an inverse relationship(Pearson correlation r =-0.655,P < 0.01).Conclusions:miR-142-5p was increased and SOCS1 was decreased in active UC patients.The expression of miR-142-5p is associated with severity of UC.There is a negative correlation between miR-142-5p and SOCS1.Part two Target gene prediction and identification of miR-142-5pObjective:To identify SOCS1 as a target gene of miR-142-5p and to investigate the effect of miR-142-5p on the expression of SOCS1 in intestinal epithelial cells.Methods:The potential gene target of miR-142-5p was predicted by miRTar Base,Target Scan and miRDB.Target 3’UTR Dual luciferase reporter wild type and its mutant vector were structured by Target Scan and Pic Tar,and transfected to TNF-α treated HT-29 cells with miR-142-5p mimic/inhibitor.The effect of miR-142-5p on target was evaluated by luciferase reporter gene assay.miR-142-5p mimic/inhibitor were transfected to TNF-α treated HT-29 cells,and expression of target was evaluated by Western Blot.Results:SOCS1 was confirmed to be the target gene of miR-142-5p through bioinformatics prediction.Bioinformatic analysis identified sequence complementarity of miR-142-5p with the 3’UTR of SOCS1.Transfection of miR-142-5p mimic/inhibitor,respectively suppressed or increased SOCS1 3’UTR(wild type)luciferase activity(1.01±0.01 vs 0.49±0.12,P<0.05;1.02±0.01 vs 1.54±0.21,P<0.05),but had no effects on SOCS1 3’UTR(mutant)(1.01±0.01 vs 0.95±0.17,P>0.05;1.01±0.01 vs 0.99±0.20,P>0.05).The expression of SOCS1 was reduced after transfection of miR-142-5p mimic(1.05±0.12 vs 0.39±0.13,P<0.05),and increased after transfection of miR-142-5p inhibitor(0.99±0.09 vs 1.42±0.23,P<0.05).Conclusions:SOCS1 was identified as a direct target gene of miR-142-5p and was inhibited by miR-142-5p.Part three miR-142-5p is involved in the pathogenesis of intestinal inflammation by targeting SOCS1Objective:To evaluate the role of miR-142-5p regulating SOCS1,and explore the regulation in the molecular mechanisms of the pathophysiology of intestinal inflammation.Methods:The expressions of miR-142-5p and SOCS1 in HT-29 cells,after treated by TNF-α,were evaluated by qRT-PCR and Western Blot.HT-29 cells treated by TNF-α was transfected with miR-142-5p mimic/inhibitor.The expressions of IL-6 and IL-8 were measured by qRT-PCR,and the concentrations of IL-6 and IL-8 were detected by ELISA.After transfection of HT-29 cells with 4 groups of SOCS1 si RNA for 48 hours,the si RNA with higher transfection efficiency was screened by qRT-PCR.The effect of SOCS1 si RNA and miR-142-5p inhibitor on IL-6 and IL-8 protein level and concentration in HT-29 cells were detected by Western Blot and ELISA.The changes of IκB-α,p IκB-α and p-p65 after transfection with miR-142-5p mimic/inhibitor were measured by Western Blot.Results:As showed by qRT-PCR,compared with 1ng/m L TNF-α(1.13±0.20)and the control group(0.97±0.13),the expression level of miR-142-5p(2.19±0.28)in HT-29 cells increased significantly after 10ng/m L TNF-α stimulationt for 1 hour,P<0.01 compared with 1ng/m L TNF-α(1.34±0.26)and the control group(1.00±0.16),the expression level of miR-142-5p(2.83±0.54)in HT-29 cells increased significantly after 10ng/m L TNF-α stimulation for 24 hours,P<0.01.Compared with 1ng/m L TNF-α(0.86±0.11)and control group(1.08±0.13),the expression level of SOCS1 in HT-29 cells(0.52±0.10)was significantly reduced after 1 hour of 10ng/m L TNF-α stimulation,P<0.01.Compared with 1ng/m L TNF-α(0.83±0.10)and control group(1.05±0.11),the expression level of SOCS1 in HT-29 cells(0.39±0.18)was significantly reduced after 10ng/m L TNF-α stimulation for 24 hours(P<0.01).Compared with the control group(1.06±0.17),the expression levels of SOCS1 in HT-29 cells after 1 hour and 24 hours of 10ng/m L TNF-α stimulation were significantly decreased(0.60±0.15,0.50±0.14),P<0.05.Compared with NC,after transfection of miR-142-5p mimic with TNF-α stimulated HT-29 cells,expression levels of IL-6(1.00±0.14 vs 1.62±0.22,P<0.05),IL-8(1.00±0.20 vs 1.66±0.22,P<0.05),and the concentrations of IL-6(187.39±48.76 vs 388.13±62.12,P<0.05),IL-8(307.18±46.35 vs 530.14±50.95,P<0.05)in cell culture medium were increased.Compared with NC,after transfection of miR-142-5p inhibitor with TNF-α stimulated HT-29 cells,expression levels of IL-6(1.04±0.13 vs 0.57±0.10,P<0.05),IL-8(1.01±0.18 vs 0.54±0.13,P<0.05),and the concentrations of IL-6(187.39±48.76 vs 388.13±62.12,P<0.05),IL-8(330.68±48.05 vs 199.22±20.70,P<0.05)in cell culture medium were decreased.The results by qRT-PCR showed that after 48 hours transfection with SOCS1 si RNA in 4 groups,the expression of SOCS1 in si SOCS1-1 group(0.39±0.09)and si SOCS1-2 group(0.35±0.06)was significantly lower than that in the control group(1.03±0.17),P<0.05,and there was no significant change in other two groups(P>0.05).The results by protein quantitative showed that the expression of SOCS1 in si SOCS1-1 group(0.39±0.11)and si SOCS1-2 group(0.36±0.11)was significantly lower than that in the control group(1.02±0.19),P<0.05.The results of Western Blot indicated that the expression of SOCS1 protein in sisocs1-1 group and sisocs1-2 group was decreased.After co-transfection of miR-142-5p inhibitor with two groups of specific SOCS1 si RNA,compared with the control group(0.96±0.15),si SOCS1-1 and si SOCS1-2 increased IL-6(1.47±0.20;1.63±0.24),P<0.05;compared with the control group(1.03±0.13),si SOCS1-1 and si SOCS1-2 increased IL-8(1.69±0.23;1.63±0.24),P<0.05;compared with the control group(211.01±37.75),IL-6 in cell culture medium was increased(335.24±46.96;356.17±52.23),P<0.05;compared with the control group(273.29±34.90),IL-8 in cell culture medium was increased(335.24±46.96;356.17±52.23),P<0.05.After transfection with miR-142-5p mimic,Western Blot showed that IκB-α was decreased and p IκB-α and p-p65 were increased.After transfection with miR-142-5p inhibitor,IκB-α was increased,p IκB-α and p-p65 were decreased.Conclusions:Inflammatory environment affected the expressions of miR-142-5p and SOCS1 in HT-29 cells,overexpression of miR-142-5p induced the production and release of inflammatory cytokines.miR-142-5p facilitated the pathogenesis of intestinal inflammation by regulating SOCS1 through the NF-κB signaling pathway. |
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