Correlation Between ADAR1 And Effects Of 8-Cl-Ado On MDA-MB-231 And SK-BR-3 Breast Cancer Cells

Objective:To investigate the correlation between ADAR1 and effects of 8-Cl-Ado on the proliferation and cell cycle of MDA-MB-231 and SK-BR-3 breast cancer cell lines.Methods:The expression of ADAR1 protein in human breast cancer tissues,adjacent tissues and different breast cancer cell lines were evaluated by WB.The effects of 8-Cl-Ado with different concentration(0?1?2?4?6?8?10?20 ?mol/L)on cell viability of MDA-MB-231 and SK-BR-3 breast cancer cell lines for 48 h were detected by MTT.WB was used to detect the expression of ADAR1 of MDA-MB-231 and SK-BR-3 breast cancer cell lines treated with 8-Cl-Ado with different concentration(0?2?4?6?8 ? 10 ? 20 ?mol/L)for 48 h.Breast cancer cells transfected with ADAR1-p110 plasmid and ADAR1-p150 plus 10 ?mol/L 8-Cl-Ado for 0?48 h,MTT was used to detect 48 h inhibition rate of every plasmid group in MDA-MB-231 and SK-BR-3 breast cancer cells.MDA-MB-231 and SK-BR-3 breast cancer cells treated with 10 ?mol/L 8-Cl-Ado for different hours respectively,the changes of the cell cycle were determined by FCM,at the same time the expression of ADAR1,P53,P21 and Cyclin D1 protein were detected by WB.After breast cancer cells transfected with ADAR1-p110 plasmid and ADAR1-p150 plasmid,the expression of ADAR1,P53,P21 and Cyclin D1 protein were detected by WB.Breast cancer cells transfected with wild-type plasmids(ADAR1-p110,ADAR1-p150)and mutant plasmids(ADAR1-?E/A,ADAR1-?R)and vector plasmid pCMV plus 10 ?mol/L 8-Cl-Ado for 0?24?48 h,each group was detected the inhibition rate by MTT.Breast cancer cells transfected with different plasmids plus 10 ?mol/L 8-Cl-Ado treated for 48 h,the changes of cell cycle were determined by FCM and migration rate detected by wound healing assay.Results:1.The expression of ADAR1-p110 and ADAR1-p150 protein in human breast cancer tissues were significantly higher than those in adjacent tissues.The data also show the higher level of ADAR1 protein expression in MDA-MB-231 and SK-BR-3 breast cancer cell lines.2.Treated with 10 ?mol/L 8-Cl-Ado could significantly inhibit cell viability of MDA-MB-231 and SK-BR-3 breast cancer cell lines.The expression of ADAR1 decreased with increasing the concentration of 8-Cl-Ado.Transfected with ADAR1-p110 plasmid and ADAR1-p150 plasmid later,the inhibition rate of 8-Cl-Ado on breast cancer cells was significantly reduced.3.After 10 ?mol/L 8-Cl-Ado treatment with MDA-MB-231 and SK-BR-3 breast cancer cells at different time,the percentage of G1 phase was increased with time,the expression of P53 and P21 protein gradually increased,the expression of ADAR1 and Cyclin D1 protein gradually decreased.Transfected with ADAR1-p110 plasmid and ADAR1-p150 plasmid later,the expression of P53 and P21 protein obviously decreased,the expression of ADAR1 and Cyclin D1 protein obviously increased.4.The inhibition rate and the G1 phase percentage of wild-type ADAR1 plasmid groups and the mutant ADAR1-?E/A group were significantly lower than those of the control group and pCMV empty vector plasmid group,the mutant ADAR1-?R plasmid group had no obvious difference.The migration rate of wild-type ADAR1 plasmid groups and the mutant ADAR1-?E/A group were obviously higher than those of the control group and the pCMV empty vector group,the mutation type ADAR1-?R plasmid group had no significant difference.Conclusion:1.The high expression of ADAR1 protein in human breast cancer tissues and breast cancer cell lines may play an important role in breast cancer process.2.Down-regulation effect of ADAR1 protein may be involved in the process of 10 ?mol/L 8-Cl-Ado inhibiting the proliferation and resulting in the G1 phase arrest of MDA-MB-231 and SK-BR-3 breast cancer cells.3.The ds-RBD region of ADAR1 may be related to the effect of 8-Cl-Ado on the proliferation inhibition and G1 phase arrest of MDA-MB-231 and SK-BR-3 breast cancer cells.