Protective Effect Of BRCA1-p21-cell Cycle Axis On Radioactive Heart Damage By Cardiac Vascular Endothelial Cells And The Mechanism Research

Objective:Detecting heart vascular endothelial cells BRCA1,p21 protein expression in radiation-induced mice heart injury model.And observing the expression of BRCA1,p21,cyclins and pRb after silence and over expression BRCA1 in human umbilical vein endothelial cells(HUVEC).And exploring the effects of BRCA1 expression level both on the cell proliferation and cell cycle,discovering the possibile protective function of "BRCA1-p21-cell cycle" axis in the radiation-induced heart disease(RIHD)from histology and molecular level.Method:The expression of BRCA1 and p21 protein in the cardiac vascular endothelial cells in radiation-induced heart injury mouse model were detected by immunohistochemistry.As for the experiment in vitro,expression plasmid vectors(pSin-EF2-BRCA1,pCDEF-p21-HA,pCDEF-BRCA1-Flag)and specific small interfering RNA(siRNA samll interfering RNA)silencing vectors were designed and synthesized.The effectiveness of the vectors were validated by sequencing and Q-PCR,and select the most effective siRNA fragment for subsequent experiments.Human umbilical vein endothelial cells were cultured in vitro.The experiment was divided into four groups including over expression negative control group(vector),pSin-EF2-BRCA1 group,interference negative control group(NC-RNAi),and siRNA-BRCA1 group.Western Blot and Q-PCR were used to detect the changes of BRCA1 and p21 in the protein and mRNA levels after transfection,and the expression of cyclinD1 and cyclin cyclinE,p-Rb,cyclinA.Cell proliferation was detected by MTT assay and BRDU staining,and cell cycle was detected by flow cytometry.The interaction of BRCA1 and p21 protein was verified by immunoprecipitation.Result:The immunohistochemistry results showed that the expression of BRCA1 and p21 protein in irradiated group was significantly higher than that in the non irradiated group.And western blot results showed that the protein and mRNA levels of BRCA1 and p21 were increased in pSin-EF2-BRCA1 group,and cyclinA,cyclinD1,cyclinE,p-Rb expression were decreased;however,the protein and mRNA levels of BRCA1 and p21 were decreased in siRNA-BRCA1 group,and cyclinA,cyclinD1,cyclinE,p-Rb expression were increased.MTT assay showed that the proliferation of HUVEC in the pSin-EF2-BRCA1 group was lower than that of the negative control group,while that was faster in the siRNA-BRCA1 group than that of the control group.BRDU staining showed that the cells in S phase in pSin-EF2-BRCA1 group was significantly lower than that of negative control group,and S phase cells were increased in siRNA-BRCA1 group.The cells were blocked in the G1 phase after overexpression BRCA1,while the siRNA-BRCA1 group was blocked in the S phase.BRCA1 and p21 were able to form agarose bead antigen antibody complexes by immunoprecipitation.Conclusion:BRCA1 is involved in cell proliferation and inhibits the proliferation of HUVEC.And BRCA1 can interact with p21 that may play a protective role in radiation induced heart disease by affecting the process of the cell cycle.