Influence Of CA12-siRNA On The Apoptosis And Chemical Resistance Of Paclitaxel-resistant Breast Cancer Cell Line MCF-7 TaxR

Objectives:This project is to explore the correlations between CA12 and the genesis and development of paclitaxel-resistant cells.Meanwhile,the effects that CA12-siRNA has on the apoptosis of breast cancer cells as well as the paclitaxel sensibility were observed.Therefore,this study shed a light to new strategies toward the paclitaxel-resistant breast cancer therapy.Methods:Normal mammary glandular cells(MCF-10),breast cancer cells(MCF-7)and paclitaxel-resistant breast cancer cells(MCF-7 TaxR)were cultured in vitro.MCF-7 and MCF-7 TaxR were divided into three groups,Experimental Group(CA12-siRNA),Negative Control Group(Scramble-siRNA)and Blank Control Group(DMSO).1.Western blotting was performed to assess the protein levels of CA12 in MCF-10,MCF-7 and MCF-7 TaxR before and after siRNA transfection.2.MTT assay was performed to detect the sensibility of MCF-7 TaxR to paclitaxel before and after the CA12-siRNA transfection.3.Phase contrast microscope,fluorescence inverted phase contrast microscope(following Hoechst 33342 staining)and flow cytometry were used to detect the apoptosis of MCF-7 and MCF-7 TaxR after the treatment with simplex siRNA transfection and the combined application of siRNA and paclitaxel respectively.4.Western blot was performed to detect the protein levels of apoptosis-related proteins mediated by mitochondrial pathway.Results:1.Compared with MCF-10 group,the expression levels of CA12 in MCF-7 and MCF-7 TaxR groups were increased.The inter-group pairwise comparisons were statistically significant in differences(P<0.01).2.After CA12 gene knockdown by siRNA,the expression levels of CA12 were decreased in the experimental groups,MCF-/ana MCF-/TaxR.it was statistically significant compared with the two control groups(the blank and negative groups)(P<0.05).3.After addition of paclitaxel to the CA12 knockdown cells,the MCF-7 TaxR experimental group showed reduced sensibility to paclitaxel compared with the two control groups(the blank and negative groups).The results were statistically significant(P<0.05).4.After addition of paclitaxel to the CA12 knockdown cells,the experimental group rose in apoptosis quantity compared with the two control groups(the blank and negative groups).The results were statistically significant(P<0.05).5.After addition of paclitaxel to the CA12 knockdown cells,western blot was adopted to detect the expressions of proteins related to mitochondrial pathway.According to MCF-7 TaxR results,the expression levels of the anti-apoptotic proteins,Bcl-2 and Bcl-xl,fell significantly compared with that of the two control groups(the blank and negative groups);however,the expression levels of pro-apoptotic proteins,Bax and Bid,increased significantly;meanwhile,the expression levels of the downstream effector molecules,Caspase-9 and caspase-7,and the activated proteins of PARP,Cleaved caspase-7,Cleaved caspase-9 and Cleaved PARP,also rose significantly.There was statistically significant difference(P<0.05).Conclusions:CA12 can be highly expressed in MCF-7 and paclitaxel-resistant MCF-7 TaxR.This is evidence that CA12 plays a part in the development of breast cancer.The CA12 expression can be knocked down by CA12-siRNA.By silencing CA12,the application of paclitaxel is able to activate the mitochondrial apoptosis pathway,promote MCF-7 TaxR apoptosis and increase the sensibility of MCF-7 TaxR to paclitaxel.