Effects Of PCNA And 14-3-3γ On Proliferation And Apoptosis In Vascular Smooth Muscle Cells

Objective: To investigate the effects of PCNA and 14-3-3γ on the proliferation and apoptosis of vascular smooth muscle cellsMethods:(1)overexpression plasmid and small interfering RNA(si RNA)of PCNA and 14-3-3γ were constructed,then transfected into rat aortic vascular smooth muscle cells(A7r5 cells),the target protein was detected by fluorescence microscopy observation of fluorescent tagged protein(GFP)and western blot was used to detect transfection efficiency.(2)After overexpression or knockdown of PCNA,the expression of cyclin D1,Bax and Bcl-2 were detected by Western blotting;the proportion of proliferating cells was detected by EDU assay;cell viability and cell apoptosis were detected by CCK-8 and flow cytometry.(3)After overexpression or knockdown of 14-3-3γ,Western blotting was used to detect the expression of cyclin D1,Bax and Bcl-2;EDU was used to detect the proportion of proliferating cells;CCK-8 and flow cytometry were used to detect cell viability and cell apoptosis.(4)Cell cycle detection by flow cytometry after knockdown of PCNA or 14-3-3γ.Result:(1)After transfected with overexpression plasmid in A7r5 cells,there is no significant difference of transfection efficiency at 24 h or 48h;si RNA was transfected using concentration gradient,the result indicated that the higher transfection efficiency and better cell status was 5ng/μl concentration.(2)Compared with the negative control group,the expression of cyclin D1 was significantly increased after overexpression of PCNA,while the expression of Bax and Bcl-2 did not change significantly;the EDU results showed that the proliferation phase cells increased in the overexpression group,while the proliferation phase cells decreased in the knockdown group.the results of CCK-8 and flow cytometry experiments showed that overexpression of PCNA promoted the proliferation of vascular smooth muscle cells,while the knockdown group inhibited the proliferation of vascular smooth muscle cells.The results of CCK-8 and flow cytometry experiments showed that overexpression of PCNA promoted the proliferation of vascular smooth muscle cells,while the knockdown group inhibited the proliferation of vascular smooth muscle cells.(3)On the other hand,the expression of Bax and Bcl-2 did not change significantly after knockdown and overexpression of 14-3-3γ,while the expression of cyclin D1 protein increased in the overexpression group;the EDU results showed that the proliferation phase cells increased in the overexpression group,while the proliferation phase cells did not change significantly in the knockdown group.the results of CCK-8 and flow cytometry experiments showed that overexpression of14-3-3γ could inhibit apoptosis of vascular smooth muscle cells,while the knockdown group promoted apoptosis of vascular smooth muscle cells.(4)After knockdown of PCNA or 14-3-3γ,the percentage of cells in S phase decreased and the percentage of cells in G0/G1 phase increased compared to controls,indicating that the cell cycle was arrested.Conclusion: PCNA can promote the proliferation of vascular smooth muscle cells,and 14-3-3γ protein has an inhibitory effect on apoptosis of vascular smooth muscle cells,which can be used as the basis for the next study.