The Influence And Mechanism Of MiRNA-21Alleviating3T3-L1Adipocytes Model Insulin Resistance

Objective:To induce insulin-resistance(IR),3T3-L1adipocyte lines were treated with high glucose and high insulin. To observe the expression of miR-21in IR adipocytes, and the influence of the over-expression of miR-21on alleviating IR. To explore the part molecular mechanisms of miR-21in improving the insulin sensitivity of adipocytes.Method:(1)3T3-L1preadipocytes were differentiated into mature adipocytes thought classic cocktail method.(2)3T3-L1mature adipocytes were divided into four groups: normal-glucose and low-insulin(NGLI) group, normal-glucose and high-insulin(NGHI) group, high-glucose and low-insulin(HGLI) group, and high-glucose and high-insulin(HGHI) group.Insulin-stimulated glucose uptake was determined by2-3H-Deoxyglucose uptake assay to estimate the insulin sensitivity.3T3-L1mature adipocytes were treated with high glucose and high insulin to establish IR model.(3) IR adipocytes were divided into three groups:IR group, adipocytes transfected with pre-miR-21plasmids(pmiR-21) group, adipocytes transfected with negative plasmids control(pNC) group, and set control group(normal adipocytes).The expression levels of miR-21in all groups were measured by using quantitative real-time RT-PCR (qRT-PCR).(4) Insulin-induced glucose uptakes of all above groups were measured by2-Deoxyglucose transport assay. (5) The protein expression levels of PTEN, Akt and phospho-Akt (Ser473)in all groups were detected by western blotting assay.Result:(1)3T3-L1preadipocytes were induced to differentiate into mature adipocyte thought classic cocktail method. Mature adipocytes appeared within10days of differentiation. Day10, adipocytes stained by Oil Red O were round or oval, and a large number of red lipid droplets can be seen gathering in the cytoplasm of mature3T3-L1adipocytes.(2) Compared to NGLI group, the insulin-stimulated glucose uptake of HGLI group3T3-L1adipocytes was decreased(P<0.05). Compared to NGHI group, the insulin-stimulated glucose uptake of HGHI group was decreased(P<0.05). Compared to NGLI group, the insulin-stimulated glucose uptake of HGHI group was drastically decreased(P<0.01).(3) Compared to control group adipocytes, the expression of miR-21in IR group was decreased (P<0.05). There was no significant difference in the quantity of miR-21between control group and pmiR-21group(P>0.05).Compared to IR group, the expression of miR-21in pmiR-21group was increased(P<0.05). Compared to control group, the expression of miR-21in pNC group was decreased(P<0.05).And there was no significant difference in the expression level of miR-21between IR group and pNC group(P>0.05).(4) Compared to control group, the insulin-stimulated glucose uptake in IR group was decreased (P<0.05). There was no significant difference in the insulin-stimulated glucose uptake between control group and pmiR-21group(P>0.05). Compared to IR group, the insulin-stimulated glucose uptake in pmiR-21group was increased(P< 0.05). Compared to control group, the insulin-stimulated glucose uptake in pNC group was decreased (P<0.05). And compared with pmiR-21group, the insulin-stimulated glucose uptake of pNC group was decreased(P<0.05).There was no significant difference in the insulin-stimulated glucose uptake between IR group and pNC group(P>0.05).(5) Compared to control group, the expression of PTEN protein in IR group was increased (P<0.05), while that of p-AktSer473was decreased(P<0.05). There was no significant difference in the expressions of PTEN and p-AktSer473between control group and pmiR-21group(P>0.05).Compared to IR group, the expression of PTEN in pmiR-21group was decreased(P<0.05), while that of p-AktSer473was increased (P<0.05). Compared to control group, the expression of PTEN protein in pNC group was increased (P<0.05), while that of p-AktSer473was decreased(P<0.05). There was no significant difference in the expressions of PTEN and p-AktSer473in all groups(P>0.05). And there was no significant difference in the expressions of Akt in all groups(P>0.05).Conclusion:(1)3T3-L1adipocytes were induced with high glucose and high insulin. The expression level of miR-21in IR adipocytes was decreased, while the expression of PTEN was increased, that of p-AktSer473was decreased.And there was no difference in the expressions of Akt.(2) Over-expression of miR-21in3T3-L1IR adipocytes transfected with pmiR-21plasmids can decrease the protein expression of PTEN and increase the phosphorylation of AktScr473, so that it can improve the insulin sensitivity of3T3-L1adipocytes.(3) MiR-21increased the Phosphorylation of AktSer473, by negatively regulating the protein expression of PTEN, so that played a role in alleviating IR.