Effect Of S100A14-siRNA On Apoptosis And Drug Resistance In Breast Cancer Paclitaxel-resistant Cell Line MCF-7 TaxR

Objective:To investigate the effect of S100A14-siRNA on apoptosis and drug resistance of Paclitaxel-resistant cell line MCF-7 TaxR in breast cancer,artificially synthesized S100A14-siRNA was used to interfere with the expression of S100A14 gene in breast cancer paclitaxel-resistant cell line MCR-7 TaxR so as to provide a new idea for the gene therapy of paclitaxel resistance in neoadjuvant chemotherapy.Methods:The normal breast cancer cells MCF-7 and breast cancer paclitaxel resistant cells MCF-7 TaxR were cultured in vitro and divided into three groups respectively:(1)blank control group(DMSO);(2)negative control group(Scramble-siRNA);(3)experimental group(S100A14-siRNA).1.The protein expression levels of S100A14 in normal mammary cells MCF-10,normal breast cancer cells MCF-7 and breast cancer paclitaxel resistant cells MCF-7 TaxR were determined by western blot method.2.The protein expression levels of S100A14 in MCF-10,MCF-7 and MCF-7 TaxR were determined by western blot method after transient transfection with S100A14-siRNA.3.After transfection with siRNA,MCF-7 TaxR cells were cultured with different concentrations of paclitaxel for 48 hours,then the changes in susceptibility to paclitaxel were determined by methyl thiazolyl tetrazolium(MTT).4.After transfection with siRNA,MCF-7 TaxR cells were cultured with paclitaxel(concentration in 3 n M)for 48 hours,then the apoptosis phenomenon was detected by inverted phase contrast microscope,Hoechst 33342 fluorescent staining and flow cytometry(Annexin V-FITC/PI staining).5.After transfection with siRNA,MCF-7 TaxR cells were cultured with paclitaxel(concentration in 3 nM)for 48 hours,then the expression levels of mitochondrial apoptosis-related proteins were determined by western blot method.Results:1.Western blot analysis showed that the expressions of S100A14 protein in the paclitaxel resistant cell line MCF-7 TaxR were significantly higher than those in paclitaxel sensitive cells MCF-7 and normal breast cells MCF-10(P<0.05).2.After transfection with S100A14-siRNA,MCF-7 TaxR cells were cultured with paclitaxel(concentration in 3 nM)for 48 hours,and then western blot analysis showed that the expressions of S100A14 protein in the experimental group were significantly lower than those in the blank control group and the negative control group(P<0.01).3.After transfection with siRNA,MCF-7 TaxR cells were cultured with different concentrations of paclitaxel for 48 hours,and then MTT assay showed that the cell viabilities of MCF-7 TaxR in negative control group and experimental group decreased with the increase of paclitaxel concentration,but the cell viabilities of the experimental group were significantly lower than that in the negative control group at the same paclitaxel concentration(P<0.05).4.After transfection with siRNA,MCF-7 TaxR cells were cultured with paclitaxel(concentration in 3 nM)for 48 hours,and then inverted phase contrast microscope observation showed that a large number of cells died;Hoechst 33342 fluorescent staining observation showed a small amount of nuclear debris with high agglutination shrink and bright blue dyeing;The flow cytometry results showed that the prophase apoptosis rates of MCF-7 TaxR in blank control group,negative control group and experimental group were(9.11± 2.01)%,(11.45 ± 1.09)%,(13.14 ± 1.87)% respectively,the advanced apoptosis rates were(8.01 ± 1.63)%,(7.54 ± 0.99)%,(72.12 ± 2.47)%respectively,and the total apoptosis rates were(17.12 ± 3.67)%,(18.99 ±2.29)% and(85.26 ± 5.01)% respectively.The prophase,advanced,and total apoptosis rates of the experimental group were significantly higher than those of the control groups(P<0.05).5.After transfection with siRNA,MCF-7 TaxR cells were cultured with paclitaxel(concentration in 3 nM)for 48 hours,and then western blot analysis showed that compared with two control groups,the expressions of Bcl-2 andBcl-xl protein in the experimental group were down-regulated,the expressions of Bid,Bax,cleaved caspase-9,cleaved caspase-7 and cleaved PARP protein were significantly up-regulated(P<0.05).Conclusions:1.The high expression of S100A14 was associated with paclitaxel resistance in breast cancer.2.S100A14-siRNA could effectively transfect the paclitaxel resistant cell line MCF-7 TaxR and silence the expression of S100A14.3.Silencing S100A14 gene could increase the sensitivity of paclitaxel chemotherapy in paclitaxel resistant cell line MCF-7 TaxR.4.Silencing S100A14 gene and combining the culture with paclitaxel could induce apoptosis by mitochondrial apoptotic pathway in paclitaxel resistant cell line MCF-7 Tax R.