Study On The Role And Mechanism Of MiR-124 In Type 2 Diabetes Mellitus And Islet β Cells

Background:In recent years,the incidence of diabetes in China has increased year by year,and tends to be younger.It is an important research goal for researchers to further study the pathological mechanism of diabetes,prevent the progression of diabetes,and develop new drugs for treatment of diabetes.miR-124 is a highly conserved and tissue-specific miRNA.Some studies have found that miR-124 is related to pancreatic tissue development and insulin secretion,which is speculated to be related to the occurrence and development of type 2 diabetes mellitus.However,there are few reports on the role and mechanism of miR-124 in type 2 diabetes mellitus.Enhancer of zeste homolog 2(EZH2)is a well-known histone modifying protein and participates in the regulation and transcription of many genes through chromatin remodeling,nucleosome modification and interaction with other transcription factors.Studies have shown that up-regulating EZH2 gene,the aging of islet beta cells can be delayed and the decrease of their proliferation ability can be inhibited.Therefore,it is speculated that EZH2 expression may be involved in the regulation of islet cell proliferation and insulin secretion,and play an important role in the development of type 2 diabetes mellitus.PartⅠ.Expression and significance of miR-124 in peripheral blood of type 2 diabetic patientsObjective To detect the expression of miR-124 in peripheral blood of patients with type 2 diabetes mellitus(T2DM)and analyze its correlation with clinicopathological parameters.Methods The clinical data of 128 patients with T2DM and 60 healthy subjects were collected.The fasting peripheral venous blood of all subjects was collected.The total cholesterol(TC),triglyceride(TG),high density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C)in peripheral blood were detected by automatic biochemical analyzer;Fasting bollod glucose(FPG)was measured by glucose oxidase and fasting insulin(FIns)were detected by chemiluminescence.The insulin resistance index(HOMA-IR)and islet β cell function index(HOMA-β)were calculated.The expression level of miR-124 in serum was detected the fluorescence quantitative polymerase chain reaction(qRT-PCR).Pearson correlation was used to analyze the correlation between miR-124 and clinical data and biochemical indexes.Results The BMI of the case group was significantly higher than that of the control group.Compared with the control group,the levels of FPG,TG,LDL-C and HOMA-IR in the case group were significantly increased(P<0.05),while the levels of TC,HDL-C and HOMA-β were significantly decreased(P<0.05).The level of miR-124 in peripheral blood of T2DM patients was significantly higher than that of healthy controls(P<0.05).The expression of miR-124 in peripheral blood of T2DM patients was significantly positively correlated with the family history,diabetic complications,and the levels of FPG,FIns and HOMA-IR,and negatively correlated with the levels of HOMA-β(P<0.05).Conclusions 1.Compared with the control group,the level of miR-124 in peripheral blood of T2DM patients was significantly increased.2.In T2DM patients,the expression of miR-124 was positively correlated with the levels of FPG,FIns,HbAlc and HOMA-IR,and negatively correlated with the levels of HOMA-β.PartⅡ.Expression of miR-124 in peripheral blood and pancreatic tissue of T2DM miceObjective To detect the pathological changes of pancreatic tissue and the expression of miR-124 in T2DM mice.Methods Mice were induced by high sugar and high-fat food combined with low-dose injection of streptozotocin(STZ)to establish T2DM mice model(DM group).Fasting FPG and FIns were measured,and HOMA-IR and HOMA-β were calculated.The pathological changes of pancreatic tissue were observed by HE staining.Apoptosis of pancreatic cells was detected by TUNEL staining.QRT-PCR was used to detect the expression of miR-124 in serum and pancreatic tissue.Western blot assay was used to detect the expression of apoptosis related proteins in pancreatic tissue.Results The levels of FPG and HOMA-IR in DM group were significantly higher than the NC group,while the level of HOMA-β was significantly lower than the NC group(P<0.05),and the level of FIns showed no significant difference between the two groups(P>0.05).The islets of rats in the NC group were without obvious pathological changes,while the islets in DM group were atrophic,the islet cells were reduced,the cells were irregular,the boundary was fuzzy,the arrangement was disordered,some islet cells were swollen and the cytoplasm was loose.The apoptosis index of DM group was(17.10±1.44)%,significantly higher than the NC group(9.02 ± 0.98)%,(P<0.05).And the expression of Caspase-3 and Bax protein significantly increased,and Bcl-2 significantly decreased,compared with NC group(P<0.05);Compared with the NC group,the level of miR-124 in serum and pancreatic tissue of DM group was significantly increased(P<0.05).And the level of miR-124 in serum was significantly positively correlated with the expression level of miR-124 in pancreatic tissue.Conclusions 1.The pancreatic tissue of T2DM model mice was obviously damaged,and a large number of cells were apoptotic,showing obvious insulin resistance.2.The expression of miR-124 in serum and pancreas of T2DM model rats were significantly increased,and there was a positive correlation between them.Part Ⅲ Effect of miR-124 on Min6 cells by targeting EZH2Objective To investigate the effect of miR-124 on the biological behavior of mouse islet β-cell Min6 and its possible regulatory mechanism.Methods Min6 mouse islet β cells were cultured in vitro and randomly divided into 4 groups:control group(BC group),high glucose treatment group(HG group),high glucose treatment+miR-124 inhibitor group(miR-124 antagomir group),high glucose treatment+miR-124 inhibitor control group(miR-124 ant-NC).The cells in HG group were cultured in high glucose(33.3 mM)complete medium.The cells of miR-124 antagomir group and miR-124 ant-NC group were cultured in high glucose complete medium and transfected with miR-124 antagomir and miR-124 ant-NC respectively.After 48 h,the expression of miR-124 was detected by qRT-PCR.ELISA was used to detect the changes of insulin secretion level in each transfected group under different concentration glucose stimulation.The proliferation ability,cell cycle distribution and apoptosis in each group were detected by CCK8 test and flow cytometry.Western blot was used to detect the expression of EZH2,Bax,Bcl-2 and Caspase-3.The target genes of miR-124 were verified by double luciferase reporter gene assay and the targeted regulation effect of miR-124 on EZH2 was analyzed.miR-124 Antagomir and siEZH2 or negative control(si-NC)were co-transfected into Min6 cells,respectively.The cells were cultured in high glucose medium for 48h.Then the changes of insulin secretion,proliferation,cell cycle and apoptosis were detected by ELISA,CCK8 assay and flow cytometry,respectively.Results Compared with BC group,the level of miR-124 in HG group was significantly increased(P<0.05).Compared with HG group,the expression level of miR-124 significantly decreased in miR-124 antagomir group(P<0.05).When stimulated by high glucose,the insulin secretion level of HG group was significantly lower than that of BC group,while the insulin secretion of Min6 cells were promoted by miR-124 antagomir(P<0.05).Compared with BC group,the proliferation activity of cells in HG group decreased significantly,but the cell proliferation activity of Min6 cells were promoted by miR-124 antagomir(P<0.05).Compared with BC group,the proportion of G0/G1 phase cells in HG group was significantly increased,and the proportion of S-phase cells was significantly decreased(P<0.05).Compared with HG group and miR-124 ant-NC group,the proportion of G0/G1 phase cells in miR-124 antagomir group was significantly decreased(P<0.05),and the number of S-phase cells was significantly increased(P<0.01).Compared with BC group,the apoptosis rate in HG group was significantly increased(P<0.05),and the expression of Bcl-2 protein was significantly decreased,while the expression of Bax and Caspase-3 protein was significantly increased;The apoptosis rate of miR-124 antagomir group was significantly lower than that of HG group(P<0.05),the expression of Bcl-2 protein was significantly increased,while the expression of Bax and Caspase-3 protein was significantly decreased(P<0.05).EZH2 was a target gene of miR-124 and negatively regulated by miR-124.The expression level of EZH2 in miR-124 antagomir group was significantly higher than that in HG group(P<0.05).Compared with miR-124 antagomir group,the expression level of EZH2 in miR-124 antagomir+siEZH2 group was significantly decreased(P<0.05).Compared with miR-124 antagomir group,insulin secretion level of miR-124 antagomir+siEZH2 group was significantly lower than that of miR-124 antagomir group and miR-124 antagomir+siNC group(P<0.05).Compared with miR-124 antagomir group,the proliferation activity was significantly decreased,the cells was blocked at G0/G1 phase,and the apoptosis rate was significantly increased in miR-124 antagomir+siEZH2 group(P<0.05).Conclusions 1.The expression of miR-124 was up-regulated in Min6 cells cultured in high glucose complete medium.2.miR-124 can target and negatively regulate EZH2 gene expression.3.miR-124 could inhibit insulin secretion in Min6 cells.4.miR-124 could inhibit the proliferation of Min6 cells.5.miR-124 could block the Min6 cells at G0/G1 phase.6.miR-124 could induce the apoptosis of Min6 cells.7.miR-124 negatively targeted the expression of EZH2 to regulate insulin secretion,proliferation,cell cycle and apoptosis of mice β-islet beta cells Min6.