The Role And Mechanism Of MiR-874-3p In The Improvement Of Hepatic Insulin Sensitivity After Sleeve Gastrectomy

ObjectiveObesity and diabetes mellitus,as two widely prevalent chronic diseases,are closely related and extremely harmful,placing an incalculable economic and psychological burden on people all over the world.Sleeve gastrectomy(SG)can not only treat obesity,but also relieve type 2 diabetes mellitus(T2DM).The remission of T2DM is closely related to the improvement of hepatic insulin sensitivity.MicroRNA(miRNA),which were considered RNA "trash" in the past,has been shown to be involved in various biological processes such as development and metabolism.However,there is a lack of researches focusing on the difference of hepatic miRNA expression and the specific mechanism of miRNA in the remission of T2DM after SG.This study intends to construct a T2DM rat model,implement SG and sham-operated surgery(SHAM),carry out miRNA sequencing,clarify the differential expressed miRNA in liver after SG,and select specific miRNA to explore its underlying mechanism in the remission of T2DM,so as to provide certain scientific clues and theoretical support for revealing the role and mechanism of miRNA in T2DM remission after SG.Methods1.T2DM rat model was established by continuous high-fat diet and intraperitoneal injection of streptozotocin,and then randomly divided into SG group(n=10)and SHAM group(n=10)to receive SG and sham-operated surgery.2.The body weight and food intake of rats were continuously monitored before and after operation.Fasting blood glucose,serum insulin,oral glucose tolerance test(OGTT),insulin tolerance test(ITT)and homeostasis model assessment of insulin resistance(HOMA-IR)were used to evaluate blood glucose homeostasis.3.At the end of the 8th week after surgery,liver samples were taken for miRNA sequencing,qRT-PCR was used to preliminarily verify the sequencing results,target miRNA was selected and its target gene was predicted.4.The target miRNA mimics and inhibitor were transfected into cells respectively.qRT-PCR and western blot were used to detect the expression of the target miRNA and the target gene.5.Dual-luciferase reporter gene assay was used to detect whether the target miRNA could directly interact with the 3’ untranslated regions(3’ UTR)of the target gene.6.After transfection of target miRNA mimics and inhibitor in cells,the changes of insulin signaling pathway related proteins were detected.7.Lentiviral vectors encoding target miRNA mimics and empty lentiviral vectors were injected into the tail vein of T2DM rats respectively.The changes of fasting blood glucose,OGTT,ITT and HOMA-IR were monitored before and after injection.The levels of target miRNA,target gene and insulin signaling pathway related proteins were detected by qRT-PCR and western blot.Results1.Body weight and food intake were similar in both groups before surgery.The food intake and body weight of SG group were significantly lower than SHAM group from the first and third week after operation,respectively.2.In SG group,fasting blood glucose,OGTT,ITT and HOMA-IR were significantly improved after operation,but no alteration in fasting serum insulin.3.There were 69 differentially expressed miRNAs between the two groups.MiR-874-3p was selected as the research object,and IKKs might be the target gene of miR-874-3p.4.Transfection of miR-874-3p mimics can increase the expression of miR-874-3p by 2300 times,and reduce the RNA and protein levels of IKKs by 67%and 74%respectively;transfection of miR-874-3p inhibitor can reduce the expression of miR-874-3p by 40%,and increase the RNA and protein levels of IKKs by 50%and 60%respectively.5.Dual-luciferase reporter gene assay showed that miR-874-3p directly targeted IKKε.6.Western blot showed that the overexpression of miR-874-3p decreased the expression of IKKs,and increased the phosphorylation levels of IRS1,PI3K and AKT;inhibition of miR-874-3p expression increased the expression of IKKs,and decreased the phosphorylation levels of IRS 1,PI3K and AKT.7.In vivo,lentiviral vectors encoding miR-874-3p mimics were injected into the tail vein of T2DM rats.Improved glucose homeostasis was found during both ITT and OGTT.MiR-874-3p levels were increased.The level of IKKs was significantly decreased,while phosphorylation levels of IRS1,PI3K and AKT were increased.Conclusions1.SG can significantly alter the hepatic miRNA expression of T2DM rats.2.MiR-874-3p can directly inhibit the expression of IKKε.3.The expression of miR-874-3p significantly increased after SG,which improved insulin resistance by inhibiting the expression of IKKε.