Effects Of BARD1 Knock-Out And Over-Expression On The Expression Of Aurora-A,BRCAD1 And P53 In Human Eukaryotic Cell

Objective:Breast cancer susceptibility gene BRCA1-associated RING domain1(BARD1)is associated with tumorigenesis,but the mechanism is unclear.The study first constructed the BARD1 knockout cell model and BARD1 over expression cell model on eukaryotic cell HEK293 T,and then investigated the influence of knocking out the BARD1 gene and over expression the BARD1 gene on BRCA1,Auror-A and P53 expression.The study will lay foundation for tumor biotherapy.Method:BARD1 knockout plasmid and BARD1 over expression plasmid were constructed.log phase human embryonic kidney epithelial HEK293 T cells were used to transfect with cationic polymer polyethyleneimine(PEI)and observed transfection efficiency under the fluorescence microscopy.Human embryonic kidney epithelial HEK293 T cells in logarithmic growth phase were seeded in six-well plates and divided into normal group(without any treatment),negative control group(transfected with empty plasmid),and knockout group(transfected with BARD1 knockout plasmid),over expression group(transfection of BARD1 over expression plasmid).The total cellular RNA and total protein were prepared.The expression of BARD1,BRCA1,p53 and Aurora-A in human embryonic kidney epithelial HEK293 T cells were detected by qPCR and Western blotting.MTT assay was used to test the cell proliferation activity.Results:The expression of BARD1 mRNA in the knockout group was significantly lower than that in the negative control group(p<0.05).The expression level of mRNA in over expression group was significantly increased(p<0.01).The expression level of BRCA1 mRNA in the knockout group was significantly higher than that in the negative control group(p<0.01).The expression level of BRCA1 mRNA in the over expression group was significantly decreased that was compared with the negative control(p<0.05).The expression level of Aurora-A mRNA in the knockout group was significantly lower than that in the negative control group(p<0.05)and the expression level of Aurora-A mRNA in the over expression group was significantly lower than that in the negative control group(p<0.05).The results of Western-blot showed that the expression of BARD1 protein in the knockout group was significantly lower than that in the negative control group(p<0.05).The BARD1 protein in the over expression group was significantly higher than the negative control group(p<0.05).The knockout group BRCA1 protein Compared with the negative control group,the expression of BRCA1 protein was significantly high(p<0.05)and the BRCA1 protein in over expression group was significantly lower than the negative control group(p<0.05).The Aurora-A protein of the knockout group was compared with the negative control group,it was significantly lower(p<0.05),the Aurora-A protein in the over expression group was significantly lower than the negative control group(p<0.05).The p53 protein in the knockout group was significantly lower than the negative control group(p<0.05).The p53 protein in the over expression group was significantly lower than that in the negative control group(p<0.05).The MTT results showed that the cell growth was significantly inhibited in the knockout group(p<0.05)and the over expression group(p<0.05)after 48 hours.Conclusions:The BARD1 knockout and over expression cell models were successfully constructed and expressed in human HEK293 T cells.The expression of downstream Aurora-A,p53 and BRCA1 proteins were affected and the cell proliferation activity also decreased when knockout BARD1 gene and over expression of BARD1 gene.The research lays a foundation for the study of BARD1 gene in tumor cells.