The Role And Mechanism Of Mir-152-3p In Insulin Resistance Of Type 2 Diabetes Mellitus

BackgroundWith the increase of aging population and obese population,the number of people with diabetes mellitus is increasing rapidly.At present,diabetes has become one of the non-infectious chronic diseases that seriously threaten human health.Diabetes is not only a major cause of blindness,kidney failure and lower limb amputations,but also a major risk factor for cardiovascular and cerebrovascular disease.A large number of studies have shown that miRNAs are closely related to the occurrence and development of type 2 diabetes mellitus(T2DM)and related complications.Therefore,further elucidating the role of miRNA in the pathogenesis of T2DM and its mechanism are helpful for the early diagnosis and treatment of T2DMObjectiveTo identify whether miR-152-3p can be used as a molecular biomarker for the early clinical diagnosis of T2DM,and to preliminarily explore the role and mechanism of miR-152-3p in insulin resistance and T2DM.Methods1.The expression of miR-152-3p in peripheral blood of healthy people and patients with T2DM was analyzedAccording to the diagnostic criteria of T2DM,the fasting peripheral blood of the healthy people and T2DM patients with no obvious complications were collected in the morning and stored for later use after anticoagulation treatment.RT-PCR was used to identify the differences in the molecular expression levels of miR-152-3p between the two groups,and the correlation between the expression levels of miR-152-3p and individual fasting glucose level or body mass index was analyzed.2.To analyze the effects of overexpressing miR-152-3p on glucose metabolism and metabolism-related gene expression in miceC57BL/6N male mice were fed with high fat diet to induce insulin resistance model and mice fed with normal diet were used as control.At weeks 12,the mice in each model were randomly divided into two groups.One group of mice were injected with adenovirus overexpressing miR-152-3p and another group of mice were injected with control adenovirus.The changes of blood glucose,glucose tolerance,pyruvate tolerance and insulin tolerance in mice were detected after overexpression of miR-152-3p.Ten days after the injection,mice were sacrificed after the fasting 12 hours and anesthetization.RT-PCR was used to detect the changes in mRNA levels of genes related to insulin signaling pathway,gluconeogenesis and fat synthesis in mouse liver.3.To analyze the effects of inhibition of miR-152-3p on glucose metabolism and metabolism-related gene expression in miceThe mice model of insulin resistance was constructed and normal mice were used as control.One group of mice were injected with adenovirus inhibiting miR-152-3p and another group were injected with control virus.The changes of blood glucose,glucose tolerance,pyruvate tolerance and insulin tolerance in mice were detected after silence of endogenous miR-152-3p.Ten days after the injection,mice were sacrificed after the fasting and anesthetization.Ten days after injection,mice were sacrificed after the fasting 12 hours and anesthetization.RT-PCR was used to detect the changes in mRNA levels of genes related to insulin signaling pathway,gluconeogenesis and fat synthesis in mouse liver.4.To screen the potential target genes of miR-152-3p by bioinformatics methodsTranscriptome sequencing analysis was performed on the liver tissues of insulin resistant mice with miR-152-3p overexpression or not to detect the changes of genes at the transcription level in liver tissues.Meanwhile,miRNA target gene prediction software was used to search potential target genes that may be regulated by miR-152-3p and related to the occurrence of diabetes.5.To analyze the regulatory effect of miR-152-3p on the expression of target gene(1)The 3'-untranslated region(3'-UTR)in mRNA of target gene was analyzed to determine the potential binding sites with the seed region of miR-152-3p.Molecular cloning was used to clone the part fragments of the 3'-UTR of the target gene into the pmirGLO dual luciferase reporter gene vector.The recombinant plasmid was transfected into HepG2 cells with miR-152-3p-agomir or NC-agomir,respectively.After cotransfection,dual luciferase gene reporting experiments were performed to detect the changes of luciferase activity of different treatment groups,thus to determine the direct regulatory effect of miR-152-3p on the target gene(2)miR-152-3p-agomir,miR-152-3p-antagomir and their corresponding negative controls were transfected into murine liver cancer cells Hepal-6,respectively.RT-PCR was used to detect the changes of target genes expression levels in cells after overexpression or inhibition of miR-152-3p.Meanwhile,cells were cotransfected with plasmid overexpressing target gene and miR-152-3p-agomir,or cotransfected with miR-152-3p-antagomir and siRNA silencing target gene,thus to verify the effect of miR-152-3p on the protein level of target gene(3)RT-PCR was used to detect the changes of target genes expression levels in the liver tissues of insulin resistant mice after overexpression and inhibition of miR-152-3pResults1.The expression level of miR-152-3p was decreased in peripheral blood of T2DM patientsCompared with healthy people,the expression level of miR-152-3p in peripheral blood of T2DM patients was significantly reduced,and it was negatively correlated with the body mass index and fasting glycemic index of individuals of the two groups.2.Effects of overexpressing miR-152-3p on glucose metabolism and metabolism-related gene expression in miceAfter the overexpression of miR-152-3p,the blood glucose level of normal mice did not change significantly,whereas the fasting blood glucose and random blood glucose levels in insulin resistant mice were lower than those in the control group.Moreover,compared with the control group,the tolerance to glucose and pyruvate,the sensitivity to insulin was all enhanced,the mRNA level of IRS2 gene in insulin signaling pathway,and mRNA level of genes related to fat synthesis,such as ACC1,were all increased,whereas the mRNA levels of gluconeogenic related genes,such as FBP1 and PCK1 were decreased in the liver of insulin resistant mice injected with adenovirus overexpressing miR-152-3p.The phosphorylation level of PI3K and GSK?3 proteins in the hepatic tissue insulin signaling pathway is increased.In contrast,the protein expression level of gluconeogenic PCK1 is decreased.3.Effects of inhibition of miR-152-3p on glucose metabolism and metabolism-related gene expression in miceAfter inhibiting the function of endogenous miR-152-3p,the glucose metabolism and sensitivity to insulin of normal mice and insulin resistant mice were decreased,and the gluconeogenesis ability was significantly enhanced.For insulin resistant mice injected with with adenovirus inhibiting miR-152-3p,the liver weight/body weight ratio decreased,the liver glycogen content did not change significantly,but the plasma insulin level increased significantly.Moreover,the mRNA level of IRS2 and ACC1 gene were decreased significantly.4.To screen the potential target genes of miR-152-3p by bioinformatics methodsThrough transcriptome sequencing analysis,we found that mRNA levels of early growth response protein 1 gene(Early growth response protein 1,EGR1)were significantly reduced in the liver of insulin resistant mice with overexpression of miR-152-3p.miRNA target gene prediction analysis also found that 3'-UTR of EGR1 mRNA contains sites with potential binding capacity to miR-152-3p5.To analyze the regulatory effect of miR-152-3p on the expression of target gene(1)The results of dual luciferase gene reporting experiments showed that overexpressing miR-152-3p could reduce the luciferase activity of the recombinant EGR1 plasmid,suggesting that mouse EGR1 was the direct target gene of miR-152-3p(2)The results of in vitro experiments showed that overexpressing of miR-152-3p could reduce the mRNA level of EGR1 gene.On the contrary,silencing endogenous miR-152-3p could increase the mRNA levels of EGR1 gene.In addition,silencing the overexpressing miR-152-3p could weaken the inhibitory effect of overexpressing miR-152-3p on the expression of EGR1 gene.Meanwhile,the results of immunoblot showed that the overexpression of miR-152-3p could decrease the protein level of EGR1.On the contrary,inhibiting the function of miR-152-3p could increase its protein level.(3)Overexpression of miR-152-3p in insulin resistant mice could significantly reduce the mRNA level of EGR1 gene in liver tissues.In contrast,silencing endogenous miR-152-3p could increase the mRNA levels of EGR1 gene in mice.Therefore,it can be determined that EGR1 is the direct target gene of miR-152-3pConclusionThe expression level of miR-152-3p decreased in peripheral blood of T2DM patients,and its expression levels was significantly negatively correlated with the body mass index and fasting glycemic index of individuals of the two groups.Downregulation of miR-152-3p expression levels may be an important indicator of insulin resistance and the occurrence of T2DM Overexpression or inhibition of miR-152-3p could improve or aggravate insulin resistance in mice by enhancing or inhibiting the expression of genes related to insulin signaling pathway miR-152-3p may play a role in inhibiting insulin resistance or the occurrence of T2DM by interfering with the expression of target gene EGR1.