The Effect Of Mitofusin2 On High Glucose On Proliferation Of Vascular Smooth Muscle Cell A7r5 And The Expression Of Genes Involved In Glucose Metabolism

Objective The incidence of diabetes mellitus complicated with macrovascular disease increased year by year, and the patients grow younger gradually. The main pathology foundation of vascular lesions is atherosclerosis.The migration of vascular smooth muscle cell to intima and the abnormal proliferation is an important part in the formation of AS. Mitofusin2(Mfn2) gene is a newly discovered proliferation inhibit gene, which can inhibits cancer cell proliferation, participates in mitochondrial fusion, regulates mitochondrial metabolism and maintains the network structure of mitochondria. The expression of Mfn2 in hypertensive rats’ aorta VSMC has decreased, therefore the upregulation of it may inhibit the process. This study was to investigate exogenous Mfn2 gene’s effects on hyperglycemia induced A7r5 cell proliferation, as well as the influence on gene expression of glucose metabolic pathway, in order to provide experimental basis for therapeutic target of Mfn2 in vascular disease.Methods The A7r5 cells were assigned into two groups based on the concentration of glucose. In the control group, the concentration was 5.5 m M, while the treatment group included the concentration of 15、25、35 and 45 m M. In each group, the cells were cultured for 72 hours and were incubated with MTT. We used ELYSA to measure the OD value in 490 nm, in order to confirm the proper glucose concentration. After culturing cells for 24h、48h、72h in the given concentration,the MTT level was measured to determine the proper culture period. the flow cytometry was used to observe the cell cycle distribution.The cells was divided into two groups: experimental group and control group,GLUT4,PPARβ,PFK,HK m RNA were detected by RT-q PCR. The cells were divided into three subgroups:control、p EGFP-N1 and p EGFP-mfn2 group. After culturing for 24 h,we detected the expression of GFP and harvested the cells after transfection of 48 h,then measured the MTT for observe the cell proliferation.We used RT-q PCR to measure the expression of GLUT4, PPARβ,PFK,HK m RNA before and after the transfection of mfn2.Results(1)MTT:After cell culturing for 72 h,we detected the MTT in A7r5 cells. The OD value in control group was 0.525±0.042、0.680±0.031、0.635±0.012,especially to 25 m M group increased obviously,the difference was significantly(P<0.05).After culturing for 24h、48h and 72 h,the MTT in experimental group was higher than the experimental group, the difference was statistically significant(P<0.05), but the MTT in 48 h and 72 h had no significant difference(P>0.05).(2) Flow cytometry analysis showed that, compared with the control group(62.8%) the proportion of cells in G1 phase in the experimental group(36.7%) decreased, the difference was significant(F=109.8, P < 0.05). Compared with the control group(23.1%) the proportion of cells in S phase in the experimental group(44.2%) increased, the difference was significant(F=103.1, P < 0.05).( 3) After successful transfection of A7r5 cells, the expression of GFP can be observed under fluorescence microscope. Compared with non-transfection group(1.105 + 0.011), MTT in p EGFP-mfn2 transfection( 0.851±0.089) decreased, the difference was statistically significant(P < 0.01).The cell proliferation in empty vector group(1.070 + 0.053) had no significant difference(P>0.05).The RT-q PCR confirmed that the GLUT4 and PPARβ decreased in the experimental group while the expression level of PFK and HK increased. while the GLUT4 and PPARβ increased in the experimental group and the expression level of PFK and HK decreased after transfection for 48 h, the difference was statistically significant.(P < 0.05).Conclusion 1. High glucose can promote cell proliferation of A7r5,and change the expression level of GLUT4, PPARβ, PFK and HK m RNA.2. Mfn2 can inhibit the influence of high glucose on A7r5 cells proliferation and regulates the expression level of GLUT4, PPARβ, PFK and HK m RNA.