Effect And Mechanism Of MiR-let-7e-5p Regulating Gas7 Expression On Apoptosis And Inflammation In Acute Pancreatitis

Objective:Acute pancreatitis(AP)is one of the most common inflammatory disorders of the digestive system characterized by high morbidity and morbidity,and the pathogenesis of pancreatitis is not well-defined.The aim of this study was to reveal the role of miR-let-7e-5p in apoptosis and inflammatory response in acute pancreatitis and its potential molecular mechanism.Methods:1.In vitro and in vivo acute pancreatitis models were constructed by intraperitoneal injection of L-arginine and caerulein respectively.MiRNAs were extracted and the relative expression levels of miR-let-7e-5p tissues and cell lines were detected by qRT-PCR.2.The miR-let-7e-5p mimic and miR-let-7e-5p inhibitor were transfected into AR42J cells,respectively,to up-regulate or down-regulate the expression level of miR-let-7e-5p in AR42J cells.CCK8,flow cytometry,Western blot(WB)and transwell were used to verify the biological characteristics of AR42J cells.3.The putative target and binding site of miR-let-7e-5p were determined using bioinformatics online software TargetScan and Starbase.A wild-type plasmid(Gas7-3’UTR WT)carrying the binding site of miR-let-7e-5p and downstream target gene Gas7 and a mutant plasmid(Gas7-3’ UTR MUT)carrying the mutation of its binding site were constructed.AR42J cells were co-transfected with miR-let-7e-5p mimic or mimic NC,respectively.The direct binding relationship between miR-let-7e-5p and Gas7 was verified by dual-luciferase reporter assay.The influence of miR-let-7e-5p on Gas7 was detected by qRT-PCR and WB.4.miR-let-7e-5p inhibitor and Gas7pcDNA3.1 were co-transfected into AR42J cells(),and the relevant biological characteristics of AR42J cells were verified by CCK8,flow cytometry,WB and transwell.Results:After the above experiments,we found that:1.The expression of miR-let-7e5p was up-regulated in acute pancreatitis tissues and AR42J cell lines.2.Inhibition of miR-let-7e-5p expression in AR42J cells induced by caerulein significantly repressed apoptosis,inflammation,and cell migration,and enhanced cell viability,while its upregulation obtained the opposite effect.3.qRT-PCR and WB results showed that upregulation of miR-let-7e-5p inhibited Gas7 expression,while knockdown promoted Gas7 expression.Dual-luciferase reporting assay confirmed that Gas7 was the direct target gene of miR-let-7e-5p and was negatively regulated by miR-let-7e-5p.4.Inhibition of miR-let-7e-5p or Gas7 overexpression significantly inhibited apoptosis,inflammatory and migration ability of cells,and enhanced cell viability.5.The results of CCK8,qRT-PCR and WB showed that Gas7 overexpression or miR-le-7e-5p knockdown could alleviat AP induced by caerulein.Conclusion:1.In the case of AP,the expression of miR-let-7e-5p was significantly increased,and miR-let-7e-5p inhibitor could reduce the apoptosis and inflammatory response and migration of pancreatic acinus cells induced by caerulein,and enhance cell vitality.However,miR-let-7e-5p mimic showed the opposite effect.2.Gas7,a target gene of miR-let-7e-5p,played an anti-inflammatory role in AR42J cells and was negatively regulated by miR-let-7e-5p.3.Gas7 overexpression or miR-let-7e-5p knockdown could alleviatAP induced by caerulein.