The Mechanism Of MiRNA-149-5p In The Pathogenesis Of Type 2 Diabetes Mellitus

BackgroundMicro-RNA(miRNA),as an endogenous non-coding small RNA,has been shown to have inclusive regulatory effects in cell.In metabolic aspects,miRNA can regulate metabolism,adipocyte differentiation,pancreatic development,beta cell differentiation,insulin biosynthesis,insulin secretion and signal transduction,and their character in metabolic diseases such as obesity and diabetes are gradually being found.As the most important metabolic disease in China,Type 2 diabetes mellitus(T2DM)accounts for 90% of the newly diagnosed diabetic patients and its prevalence tends to be younger.Therefore,it is of great value to identify new miRNAs and reveal the mechanism of action in the pathogenesis of T2 DM.PurposeTo indentify the level of miR-149-5p in peripheral blood from healthy people and T2 DM patients,to analyze the relationship between the level of miR-149-5p and clinical diabetic indexes,to screen out potential target genes of miR-149-5p through bioinformatics analysis and cellular experiments,to verify the effect of miR-149-5p on the regulation of target genes by constructing recombinant plasmids,to investigate the relationship between miR-149-5p and insulin signaling pathway,glucose metabolism pathway and lipid metabolism pathway,to elucidate the molecular mechanism of miR-149-5p in the pathogenesis of T2 DM.Methods1.Identifying the relationship between miR-149-5p level and the occurrence of T2DM(a).Collecting the fasting peripheral blood from T2 DM patients without any treatment and complications from healthy donor with similar age,reserving the blood at-80? after EDTA treatment.Meanwhile,collecting the clinical biochemical data from these two groups of people as well as the 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).Using the real-time PCR(RT-PCR)technique to identify the expression levels of miR-149-5p in two groups.miR-149-5p 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-149-5p and diabetic indexes were analyzed,involving fasting blood glucose level,triglyceride level,high density lipoprotein level,low density lipoprotein level and body-mass index.2.Identification of the target gene controlled by miR-149-5p in T2DM(a).We predicted the potential T2DM-related target genes of miR-149-5p by using the following softwares,including DIANA TOOLS,miRDB and Target Scan.(b).miRNA agomir/antagomir was transfected into HepG2 cells and NC-agomir/antagomir was used as negative control.Confirmation of miRNA and target gene expression level in human HepG2 cells was performed by RT-PCR,and the levels of miR-149-5p in cells was normalized to U6 gene.Meanwhile,levels of target genes of miR-149-5p were detected normalized to actin gene.Genes regulated significantly would be considered as the targets of miR-149-5p.(c).Analyzing the 3' untranslated region(3'UTR region)of target genes to determine their potential combining sites with miR-149-5p.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-agomir or miR-149-5p-agomir.Dual-luciferase reporter assay was carried out to test the direct regulation of miR-149-5p on its target genes after 24 hours.3.To explore the effect of miR-149-5p on target genes and insulin signaling pathway(a).The effect of miR-149-5p changes on its target genes and insulin signaling pathway was analyzed by RT-PCR.The corresponding primers were designed for the important protein molecules in the insulin signaling pathway,including AKT,IRS1 and IRS2,thus to detect how the overexpression or inhibition of miR-149-5p could change their levels in HepG2 cells.(b).The expression levels of target gene and phosphorylated AKT were detected by western blot after overexpression or inhibition of miR-149-5p.Results1).The difference of miR-149-5p level in healthy people and T2 DM patients: The RT-PCR results demonstrated that miR-149-5p level in T2 DM patients was 1.3 times higher than in healthy people,and the level of miR-149-5p was positively correlated with the level of fasting glucose and BMI index.However,it has no correlation with levels of high density lipoprotein,triglyceride,low density lipoprotein and total cholesterol.2).The screening of potential target genes of miR-149-5p.We screened out 12 potential diabetes related target genes of miR-149-5p through bioinformatics analysis.3).Effects of overexpression or inhibition of miR-149-5p on the level of target genes.The miR-149-5p level was nearly a hundred fold rise in HepG2 cells after the transfection of miR-149-5p agomir and IGFBP5 protein level was decreased.Meanwhile,the expression levels of IGFBP5 was increased after the transfection of miR-149-5p antagomir.4).Construction of recombinant plasmids.A further bioinformatics analysis showed that3'UTR regions of IGFBP5 containing potential binding sites to miR-149-5p,and DNA sequences coding 3'UTR regions of these genes were cloned into the pmirGLOdual-luciferase plasmid.The sequencing showed that IGFBP5 recombinant plasmids were successfully constructed.5).The direct regulation of miR-149-5p on its target gene.The results demonstrated that the luciferase activity was significant declined(P<0.05)when the IGFBP5 recombinant plasmid was transfected with miR-149-5p agomir in HepG2 cells,indicating the direct regulation of miR-149-5p on IGFBP5.6).To determine the effect of miR-149-5p on the transcription and expression of insulin-related genes.RT-PCR showed that the expression of Irs1,Irs2 and Akt was decreased(P <0.01)after overexpression of miR-149-5,whereas their levels were increased(P<0.01)when miR-149-5p was inhibited.Western Blot showed that the level of p-AKT in HepG2 cells was increased after treatment with miRNA149-5p antagomir(P <0.05).Conclusions1.MiR-149-5p was abnormally higher in the blood of T2 DM patients,and the miR-149-5p level was significantly correlated with multiple diabetes clinical indicators.2.IGFBP5 may be a target gene that directly regulated by miR-149-5p,and overexpression of miR-149-5p promotes the occurrence of T2 DM by inhibiting IGFBP5 expression.3.The overexpression of miR-149-5p inhibits the expression of Irs1,Irs2 and Akt in the insulin signaling pathway,whereas inhibition of miR-149-5p strengths the activity of insulin signaling pathway.