The Reserch Of MiR-146a Regulation In Stress Ulcer

Objective:Sudden intense stimulations like war,terrorist attack,natural disaster and emergency blast injury or burns,cause violent reactions of body systems that showed as stress ucler in alimentary canal with high morbidity and mortality.Its pathogenesis is mainly involve decrease of the barrier effect in the gastric macosa,excessive release of gastric acid and inflammatory mediators,bacterial and endotox translocation,helicobacter pylori infection,neuroendocrine axis inbalance,etc.,but there is no great breakthroughin its molecular mechanism.The study found that the occurrence of stress ulcer and NF-κB activation is closely related,but,The mechanism by which NF-kB signaling pathways are involved in stress ulcers is ambiguous.Our previous study found that microRNA-146a is an endogenous regulatory factor dependent on NF-kB,which is involved in the regulation of inflammatory response.In this study,we established a model of stress ulcer in vivo and in vitro,and used quantitative PCR to detect the expression of microRNA-146a in the model of stress ulcer,and used bioinformatics prediction software and dual luciferase reporter vector to identify the target gene of microRNA-146a.To study the mechanism of microRNA-146a inhibition of the development of stress ulcers,we analysed its negative regulatory function by gene transfection and PCR."Mechanism of stress ulcer occurrence,microRNA-146a changes and inhibitory effect" is of great significance to further elucidate the molecular pathogenesis of stress ulcer,and it is important to provide laboratory data for the treatment and prevention of stress ulcers in wartime.Methods:1.The expression of miR-146a in gastric mucosa was confirmed by in vivo experiment.We observed pathology by HE and detected the expression of NF-kB p65 and miR-146a by PCR in gastric mucosa of experimental group and control group.2.Establishment of stress ulcer model of human gastric adenocarcinoma AGS cells and expression of miR-146a in model.The content of malondialdehyde(MDA)and superoxide dismutase(SOD)and the expression of NF-kB p65 and microRNA-146a were measured at 6 h after treatment with 200 umol/L H2O2.3.Functional study of microRNA-146a in the mechanism of stress ulcer.The target genes of miRNAs in AGS cells were identified by bioinformatics prediction,luciferase assay and real-time quantitative PCR;we studied the mechanism of miRNAs regulating inflammatory response in stress ulcer by in vitro overexpression or inhibition of expression of miRNAs,PCR and other experiments.Results:1.The expression of miR-146a in gastric mucosa was confirmed by in vivo experiment.Animal model In the 100-fold optical microscope,compared with the control group,the gastric mucosa epithelium of the experimental group showed obvious defects and the surface covered with inflammatory exudate,indicating that the model was established successfully.Real-time PCR results showed that the microRNA-146a MRNA expression increased by a factor of 4(P<0.01)and NF-kB p65 mRNA expression increased by 2-fold(P<0.01).2.Establishment of stress ulcer model of human gastric adenocarcinoma AGS cells and expression of miR-146a in model.Compared with the control group,the content of MDA in the experimental group was significantly increased(P<0.01),SOD activity was significantly decreased(P<0.01),indicating that the cell model was stable;microRNA-146a and NF-kB p65 mRNA expression were significantly increased(P<0.01,P<0.01).3.Functional study of microRNA-146a in the mechanism of stress ulcer① Prediction and identification of microRNA-146a target gene TargetScan and Miranda predicted the potential target gene APPL1 of microRNA-146a.The luciferase reporter vector confirmed that microRNA-146a could complement the suspicious target gene APPL1 3’-UTR.Real-time PCR results showed that microRNA-146a could degrade target gene APPL1 mRNA thus inhibits its translation.② MicroRNA-146a inhibits stress-induced ulcer inflammation Overexpression of microRNA-146a significantly reduced the expression of NF-kB in the inflammatory chain of stress-induced ulcer cell model,significantly reduced the production of MDA and increased the activity of SOD,which indicated that miRNA-146a was negatively fed in the inflammatory response of stress ulcer Regulation.Conclusions1.It was proved that stress ulcer can up-regulate the expression of microRNA-146a in gastric mucosa and human gastric adenocarcinoma AGS cells,which may be involved in the development process.2.The target gene APPLl of microRNA-146a was verified,and microRNA-146a could regulate the inflammatory reaction of stress ulcer through this target gene.