| Background:Ulcerative colitis(UC)is a chronic intestinal nonspecific inflammatory disease,but its pathogenesis has not yet very clear.Statistics show that,in recent years,the morbidity of UC is increase obviously than before in our country.Most researchers think it maybe associated with the economic development,the change of the pepole diet and life-style,environment,genetic,immune factors and so on.Because of the disease recurrence and difficult to treatment,its serious impact the UC patients quality of life,and lead a lot of the medical resources waste.So it need to research the pathogenesis of UC,expect to provide certain theoretical basis for find new ways to treatment the UC patients.microRNAs(miRNAs)are a type of short sequence(a length of approximately 22 nucleotides)with non-coding,single strand small RNAs,it can regulate gene expression via complementary base pairing with the 3’-untranslated region(3’-UTR)of target mRNAs,resulting in mRNA degradation or translational suppression.Bioinformatics analysis research reports that,miRNAs are play an important role in biological processes,such as cell proliferation,differentiation,apoptosis and immunoreactions,so miRNAs may involved in regulation many signal transduction pathway.Adenosine is a kind of endogenous purine nucleosides,it can play an important role in physiological and pathological conditions,by binding specificity and activation of adenosine receptors.In physiological and pathological conditions,adenosine A2a receptor(A2aAR)mRNA and protein express in many kinds of people and animals gastrointestinal tissue and cells,and participate in regulating intestinal immune and inflammatory response.A large number of studies have found that,the expression of A2aAR was decreased when the intestinal inflammation occurred.Other research reported that,the miR-16 also express in intestinal epithelial cell,and the expression of miR-16 was increased when the intestinal inflammation occurred.Both of them express in intestinal epithelial cell,and the expression were changed when the intestinal inflammation occurred,if there is a certain correlation of them in UC has not been reported.Thus,this study was to investigate the roles and mechanisms of miR-16 and A2aAR in UC,hope it will provide new disease diagnosis and therapy by use miRNAs and A2aAR,to improve the prognosis and quality of life for UC patients.Part 1:Research on the expression of miR-16 and A2aAR and their correlation in UC tissuesObjective Our study aim to characterize the localization and distribution of the A2aAR in colon tissues,detected the expression of miR-16 and A2aAR mRNA and protein in the tissues,and analyze the correlation between the expression of miR-16 and A2aAR in UC tissues,in order to investigate the possible mechanism of miR-16 and A2aAR involved in the occurrence and development process of UC.Methods In our study,we selected 20 healthy patients tissue samples,22 IBS tissue samples,28 UC tissue samples,use the immunofluorescence(IF)to locate the A2aAR in colonic mucosa biopsies,the expression of miR-16 and A2aAR mRNA were detected by real-time quantitative PCR(qRT-PCR),and the expression of A2aAR protein were examined by western blot,moreover,we analyzed the relationship between the expression of miR-16 and A2aAR mRNA and protein in UC tissues.Results The IF results showed clear expression of the A2aAR in colonic epithelial cells.The qRT-PCR results indicated that the expression of miR-16 was significantly increased in UC tissues than normal controls and IBS tissues,and the difference was statistically significant(P<0.01),however,the expression of A2aAR mRNA was significantly deceased in UC tissues than normal controls and IBS tissues,and the difference was statistically significant(P<0.01).The western blot results showed that compared with normal controls and IBS tissues,the expression of A2aAR protein was decreased significantly in the UC tissues,and the difference was statistically significant(P<0.01).Furthermore,we find there was not obvious correlation between miR-16 and A2aAR mRNA(r = 0.095,P>0.05),but the miR-16 and A2aAR protein expression were significant negative correlation in UC tissues(r =-0.438,P<0.05).Conclusion The expression of miR-16 and A2aAR were differential in UC tissues,and there have a negative correlation with each other.miR-16 may be involved in the occurrence and development process of UC through regulate A2aAR.Part 2:Research on the miR-16 regulate the expression of A2aAR in intestinal epithelial cellsObjective We aim to detect the expression of A2aAR mRNA and protein with changed the miR-16 level,and clarified the regulation of A2aAR by miR-16.Methods The target mRNAs of miR-16 were predicted by bioinformatic analysis tools,and using a dual-luciferase reporter assay to proved A2aAR was a direct target of miR-16.Transfect different concentration of miR-16 mimics,miR-16 inhibitor and corresponding NC into intestinal epithelial cells(HT-29),using qRT-PCR to detect the expression of miR-16 and A2aAR mRNA,western blot detected the expression of A2aAR protein in each transfected cells.Results We applied Target Scan to predict putative binding sites of miR-16 and found A2aAR mRNA 3’-UTR was a binding site of miR-16.After co-transfected with the pmiR-A2aAR-wt and miR-16 mimics or miR-16 inhibitor or corresponding NC into HT-29 cells,we found that the luciferase activity was significantly decreased in miR-16 mimics when compared with the corresponding NC group,the difference was statistically significant(P<0.05),and the luciferase activity was significantly increased in miR-16 inhibitor when compared with the corresponding NC group,the difference was statistically significant(P<0.05).There was no difference between miR-16 mimics or miR-16 inhibitor and corresponding NC group when co-transfected with the pmiR-A2aAR-mut(P>0.05).After transfected different concentration(50 nM,100 nM,150 nM)of miR-16 mimics,miR-16 inhibitor and corresponding NC into HT-29 cells,compared with the corresponding NC group,the expression of miR-16 was significantly increased in miR-16 mimics group,and the expression of A2aAR mRNA and protein were significantly decreased,they changed in a concentration-dependent manner,the differences were statistically significant(P<0.05),compared with the corresponding NC group,the expression of miR-16 was significantly decreased in miR-16 inhibitor group,and the expression of A2aAR mRNA and protein were significantly increased,they changed in a concentration-dependent manner,the differences were statistically significant(P<0.05).Conclusion A2aAR is one of the target genes of miR-16,and miR-16 can negatively regulates the expression of A2aAR.Part 3:Research on the miR-16 and A2aAR effect on the related inflammatory NF-κB signaling pathwayObjective Our study aim to detect the miR-16 and A2aAR effect on the related inflanmmatory NF-κB signaling pathway.Methods We used TNF-a to stimulate the HT-29 cells to establish inflammatory model,co-transfected with the miR-16 mimics or miR-16 inhibitor or miR-16 inhibitor+A2aAR-siRNA or corresponding NC into HT-29 cells which after used TNF-a stimulated,then using western blot to detected the expression of cytoplasm and nucleus NF-κB p65 protein,qRT-PCR and ELISA detected the expression of IFN-r and IL-8 mRNA and their protein.Results After HT-29 cell stimulated with TNF-a,we find the NF-κB p65 protein translocate from the cytoplasm to the nucleus,and the downstream pro-inflammatory cytokines IFN_y and IL-8 were significantly increased.When compared with the corresponding NC group,miR-16 mimics group exhibited a significant decrease the NF-κB p65 protein in cytoplasm and an significant increase the expression of the nucleus NF-κB p65 protein,the differences were statistically significant(P<0.05),meanwhile,the expression of downstream pro-inflammatory cytokines IFN-y and IL-8 mRNA and protein also increase significantly,the differences were statistically significant(P<0.05).When compared with the corresponding NC group,miR-16 inhibitor group exhibited an significant increase the NF-κB p65 protein in cytoplasm and a significant decrease the expression of the nucleus NF-κB p65 protein,the differences were statistically significant(P<0.05),meanwhile,the expression of downstream pro-inflammatory cytokines IFN-γ and IL-8 mRNA and protein both significantly decrease,the differences were statistically significant(P<0.05).Co-transfected with miR-16 inhibitor+A2aAR-siRNA group,we find a significant decrease the NF-κB p65 protein in cytoplasm and an significant increase the expression of the nucleus NF-κB p65 protein,the differences were statistically significant(P<0.05),meanwhile,the expression of downstream pro-inflammatory cytokines IFN-y and IL-8 mRNA and protein also increase significantly,the differences were statistically significant(P<0.05)when compared with miR-16 inhibitor+siRN A-N C.Conclusion miR-16 can regulate the inflammatory responses by activating NF-κB signaling pathways,and this effects may be partly dependent on regulates the expression of A2aAR. |
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