Study On The Mechanism Of MiRNA-152 In The Pathogenesis Of Type 2 Diabetes Mellitus

BackgroundmiRNAs plays an important role in the occurrence and development of type 2 diabetes mellitus(T2DM)and diabetic complications.miRNA participates in the balance of blood sugar through regulating the differentiation of pancreatic beta cells,synthesis and secretion of insulin,and insulin signal pathway.Therefore,it is valuable to identify new miRNA and reveal its role in the development of T2 DM.PurposeTo indentify the level of miRNA-152 in peripheral blood from healthy people and T2 DM patients,to analyze the relationship between the level of miRNA-152 and clinical diabetic indexes,to screen out potential target genes of miRNA-152 through bioinformatics analysis and cellular experiments,to verify the effect of miRNA-152 on the regulation of target genes by constructing recombinant plasmids,to elucidate the molecular mechanism of miRNA-152 in the pathogenesis of T2 DM.Methods1.Identifying the relationship between miRNA-152 level and the occurrence of T2DMa)Collecting the fasting peripheral blood from healthy donor and T2 DM patients without any treatment and complications,reserving the blood at-80? after EDTA treatment.Meanwhile,we collected the blood clinical biochemical data of people in these two groups and medical records of T2 DM patients.b)Total RNA was extracted using Trizol reagent after adding external reference genes into the blood,and miRNA was modified Poly(A)tails through Plus A tail reaction,followed by reverse transcribed into cDNA.c)The real-time PCR(RT-PCR)technique was used to identify the expression levels of miR-152 in two groups.miR-152 was amplified by forward primer and universal reverse primer,external reference gene was also amplified to adjust the RT-PCR reaction.d)The relationships between the level of miR-152 and diabetic indexes were analyzed,involving fasting blood glucose level,triglyceride level,high density lipoprotein level,low density lipoprotein level,total cholesterol level,and body-mass index(BMI).2.Identification of the target gene controlled by miR-152 in T2DMa)We predicted the potential T2DM-related target genes of miRNA-152 by using the following softwares,including DIANA TOOLS,miRanda and TargetScan.b)miRNA-152 mimic was transfected into HepG2 cells and NC-mimic was used as negative control.RT-PCR was used to detect the levels of miRNA-152 in cells normalized to U6 gene.Meanwhile,levels of target genes of miRNA-152 were detected normalized to actin gene.Genes down-regulated significantly would be considered as the targets of miRNA-152.c)Analyzing the 3' untranslated region(3'UTR region)of target genes to determine their potential combining sites with miRNA-152.The 3'UTR region of target genes was cloned into pmirGLO dual-luciferase plasmid,and constructed recombinant plasmids were determined by sequencing.d)The empty plasmid and recombinant plasmids were cotransfected into HepG2 cells with NC-mimic or miRNA-152 mimic.Dual-luciferase reporter assay was carried out to test the direct regulation of miRNA-152 on its target genes after 24 hours.Results1.The difference of miRNA-152 level in healthy people and T2 DM patients.The results demonstrated that miRNA-152 level in T2 DM patients was 1.4 times higher as in healthy people,and the level of miRNA-152 was positively correlated with the level of fasting glucose,but negatively correlated with the level of high density lipoprotein.However,it has no correlation with levels of triglyceride,high density lipoprotein,total cholesterol,and BMI.2.The screening of potential target genes of miRNA-152.We screened out 8 potential target genes of miRNA-152 through bioinformatics analysis.3.Effects of overexpression of miRNA-152 on the level of target genes.The miRNA-152 level was nearly a hundred fold rise in HepG2 cells transfected with miRNA-152 mimic.Meanwhile,the expression levels of its target genes,including ADAM17,CSF1 and MET,were obviously decreased.4.Construction of recombinant plasmids.A further bioinformatics analysis showed that 3'UTR regions of CSF1 and MET containing potential binding sites to miRNA-152,and DNA sequences coding3'UTR regions of these genes were cloned into the pmirGLO dual-luciferase plasmid.The sequencingshowed that three recombinant plasmids were successfully constructed.5.The direct regulation of miRNA-152 on its target genes.The results demonstrated that the luciferase activity was significant declined when the CSF1 and MET recombinant plasmid was transfected with miRNA-152 mimic into HepG2 cells,indicating the direct regulation of miRNA-152 on these genes.Conclusions1.We found an abnormal elevated level of miRNA-152 in the blood of T2 DM,compared with that in healthy people.Moreover,and the level of miRNA-152 was positively correlated with the level of fasting glucose,but negatively correlated with the level of high density lipoprotein.2.CSF1 and MET were identified as target genes of miRNA-152 through bioinformatics analysis and in vitro experiments,suggesting that overexpression of miRNA-152 may be involved in the occurrence of T2 DM by interfering the expression of CSF1 and MET.