Relationship Between Liver Cell Microrna Expression Profile And High Fat-induced Insulin Resistance In Mice

Obesity has become a global public health problem,and its prevalence continues to rise by up to 40% in the next 10 years.It is particularly worrying that obesity can cause a range of diseases,including type 2 diabetes,cardiovascular disease,high blood pressure,chronic kidney disease and non-alcoholic fatty liver.The obesity-related insulin resistance is an early event in the process of these diseases.The pathogenesis of insulin resistance is complex,which might be associated with endoplasmic reticulum stress,inflammation and gut microbes imbalance.However,its precise molecular mechanisms are still unclear.The biological effects of insulin depend on the transduction pathway of the signal molecules in the targrt cells,so the normal expression of intracellular signal transduction molecules are particularly important.In recent years,a class of small RNA(microRNAs,miRNAs)involved in the post-transcriptional regulation of gene expression attracts the extensive attention,which plays an important role in many biological processes.mi RNAs are a class of evolutionarilyhighlyconserved small single-stranded non-coding RNA molecules of 19~22 nucleotides,which negatively regulate gene expression at the post-transcriptional level by inhibiting the translation of target genes.In 2004,Poy first found that miR-375 in pancreatic islet cell can regulate insulin secretion,inferring that it may be involved in the pathological process of type 2 diabetes.Subsequently,miRNAs as new gene expression regulator have become a research hot point in the molecular mechanism of diabetes and its complications.The liver,a majorinsulin target organ,is responsiblefor the glucose metabolism.To date,It is not clearwhether the hepatic miRNAs are involved in the insulin resistancein obesity situation.Therefore,we screened the differentially expressed miRNAsof liver tissues in mice with a high fat diet-induced insulin resistance using microRNAs microarray,and identified the expression levels of miRNAs in liver tissues and liver AML12 cells with insulin resistance using real-time fluorescence quantitative PCR(RT-qPCR).Further the target genes of differential expressed miRNAs and target genes related signaling pathway were predicted by bioinformatics to investigate the relationship between insulin resistance and differentially expressed mi RNAs.In this study,those choosed liver samples were derived from the previous insulin resistance mouse models induced by the high fat diet in our laboratory.Three samples of liver tissue were randomly selected from the control group and the insulin resistance group,respectively.The differentially expressed miRNAs were screened using microRNA microarray.To verify the accuracy of the screening results,a stem-loop reverse transcriptional real-time fluorescence quantitative PCRwas established to identify the differentially expressed miRNAs in mouse liver tissues.Furthermore,palmitic acid(PA)-inducd insulin resistance in AML12 cell model was established.The experiment was divided into blank group、control group and 4 insulin resistance groups and each group was made ofparallel 6-well using 24-well culture plateand DMEM low glucose medium.The blank group only contained DMEM low glucose medium without cells;The control group contained cells without PA;The 4 insulin resistance groups contained different concentrations of PA.The insulin resistance groups were induced with PA of 0.25,0.5,0.75,1mmol/L for 8h,respevtively,thenincubated in DMEM culture with 10-9mol/L insulin for 12 h.CCK8 method was usedto detect the proliferation of AML12 cell.Using glucose oxidase method,the glucose level in medium was measured,and then the glucose consumption counted.Accumulation of intracellular lipids was observed by Oil Red O staining.The above three methods were used to determine whether the insulin resistance cell model was successfully established.RT-qPCRwas used to identify the differentially expressed miRNAs in AML12 cells to further verify the accuracy of the microRNA microarray screening results.The target genes of differentially expressed miRNA were predicted by combined utilization of Targetscan database,miRanda database and PITA database.The GO and KEGG database molecular annotation system were used to investigate the main effects of the miRNAs-targeted genes on the biological functions or signal pathway.The STRING10.0 was used to analyze protein-protein interaction.The screening results of microRNA microarray chip showed thatcompared with control group,the expression levels of miR-1897-3p,miR-690 and miR-7a-5p detected in insulin resistance group were significantly decreased,suggesting that the mi RNAs expression profile of liver tissue was changed in mice with insulin resistance.RT-qPCR results showed thatcompared with control group,the expression levels of miR-1897-3p,miR-690 and miR-7a-5p detected in the insulin resistance group were more than 2 timesdecreased(P<0.01),being consistant with microarray chip results.The AML12 cell model of insulin resistance was successfully induced with PA.CCK8 results showed that,when PA concentration was 1 mmol/L,the proliferation rate and activity of cell were decreased,and the cell was damaged.When the concentration of PA was 0.5,0.75 and 1mmol/L,the utilization of glucose in cells was decreased,compared with contral group.With the increase of PA concentration,the consumption rate of glucose in insulin resistance group cells was significantly decreased by 49.91%,31.89%,respectively(P<0.01).The results of Oil Red O stainingshowed thatthe edge of cell were clear with a small amount of orange lipid droplets in the control group;in the PA-induced cells,the accumulation of orange lipid droplets was saw,and with the increase of PA concentration,the number and volume of lipid droplets increased gradually.Based on the comprehensive analysis of cell glucose consumption,cell proliferation rate and Oil Red O staining results,we determined the best concentration of PA was 0.75mmol/L for establishing insulin resistance model of AML12 cells.The RT-qPCR results showed that the expression levels of miR-1897-3p,miR-690 and miR-7a-5pin the insulin resistance group weremore than 2 times decreased(P<0.01),compared with control group,being consistant with microarraydetection results.Bioinformatic analysis results showed that the each differentially expressed mi RNAs can target multiplegenes,namely,miR-1897-3phaving 745 target genes,for mi R-690425 target genes,as to miR-7a-5p having 3114 target genes.GO analysis indicated most of target genes belong to protein-binding genes of molecular function and membrane protein genes of cell components.Functionally,most of the three mi RNAs-targeted genes were involved in transcription control.KEGG analysis showed the differntially expressed miRNAs targeted genes were enriched in multiple signaling pathways and a total of 16 target genes are regulated by two differentially expressed miRNAs.Among of the 16 target genes protein,8 protein have interactions with ≥3 proteins.Rac1,Rhoa,Prkcz,Tgfbr2,Itch,Ube2d3 and Epha7 protein are located in the central node of the network,and if they were removed,the network would be destroyed.Inconclusion,the liver tissue miRNAs expression profile is different in mice with a high fat diet-induced insulin resistance,and the expressionlevels of miR-1897-3p,miR-690 and miR-7a-5p are significantly decreased.The AML12 cell model of the PA-induced insulin resistance is successfully established,and the expression levels of miR-1897-3p,miR-690 and miR-7a-5p are also decreased in the cells of insulin resistance.The miR-1897-3p,miR-690 and miR-7a-5p may be involved in the physiopathologic process of insulin resistance,via affecting the normal insulin signalingcascade reaction by regulating the expression levels of Rac1,Rhoa,Prkcz,Tgfbr2,Itch and Ube2d3.The findings enrich the miRNAs differential expression profiles in insulin resistance and lay a foundation for further study on the action mechanisms of miRNAs.