In Vitro Study Of Non-homologous Terminal Junction Activity On Paclitaxel Resistance In Triple Negative Breast Cancer

Objective:To explore whether non-homologous terminal junction activity is involved in paclitaxel resistance of triple negative breast cancer by vitro cell experiments,to observe the effect of inhibiting its activity on paclitaxel-resistant cells of triple-negative breast cancer,and to explore its potential mechanism,hoping to provide a theoretical reference for solving the paclitaxel resistance in triple negative breast cancer.Methods:Firstly,triple negative breast cancer cells（MDA-MB-231）and the paclitaxel-resistant triple negative breast cancer cells（MDA-MB-231/PTX）were resuscitated and cultured in vitro.The expression of DNA ligase IV（LIG4）was detected by RT-PCR and Western blot.The relationship between the expression of LIG4 in triple negative breast cancer patients and survival prognosis was analyzed by TCGA database.The sensitivity of the two cells to different concentrations of paclitaxel and different concentrations of SCR7（LIG4 inhibitor）was compared by CCK8 method,and the sensitivity of MDA-MB-231/PTX cells to paclitaxel under the combination of the two drugs was detected by CCK8 method.The MDA-MB-231/PTX cells were divided into control group（MDA-MB-231/PTX）,paclitaxel group（MDA-MB-231/PTX+Paclitaxel）,and paclitaxel combined with SCR7 group（MDA-MB-231/PTX+Paclitaxel+SCR7）.The proliferation ability was observed by clone formation assay,the migration ability was observed by scratch assay and the invasion ability was observed by Transwell assay.The apoptosis and cell cycle changes were analyzed by Flow cytometry.The expression of LIG4,XRCC1,PARP1 and drug resistance protein P-g P in MDA-MB-231/PTX cells was detected by Western blot.Results:The results of RT-PCR and Western blot showed that the expression of LIG4 in MDA-MB-231/PTX cells was higher than that in MDA-MB-231 cells（P（27）0.05）.Bioinformatics analysis showed that triple negative breast cancer patients with high expression of LIG4 had shorter survival time than those with low expression of LIG4（P（27）0.05）.The results of CCK8 showed that the 48h IC₅₀of paclitaxel in MDA-MB-231/PTX cells was significantly higher than that in MDA-MB-231 cells（P（27）0.05）;there was no significant difference in 48h IC₅₀ between MDA-MB-231/PTX cells and MDA-MB-231 cells treated with SCR7（P（29）0.05）;compared with the48h IC₅₀ of MDA-MB-231/PTX cells treated with paclitaxel alone,the 48h IC₅₀ of MDA-MB-231/PTX cells treated with paclitaxel combined with SCR7 was significantly reduced（P（27）0.05）.Cell clone formation assay,scratch assay and Transwell assay results showed that compared with the control group and paclitaxel group,paclitaxel combined with SCR7 group significantly inhibited the proliferation,migration and invasion of MDA-MB-231/PTX cells（P（27）0.05）.Flow cytometry analysis showed that compared with other groups,paclitaxel combined with SCR7group significantly increased the apoptosis rate of MDA-MB-231/PTX cells and the aggregation of cells in G2/M phase（P（27）0.05）.Western Blot results showed that the expression of LIG4,XRCC1,PARP1 and P-g P protein in paclitaxel combined with SCR7 group was significantly lower than that in other groups（P（27）0.05）.Conclusion:1.LIG4 is highly expressed in MDA-MB-231/PTX cells,and the survival time of triple negative breast cancer patients with high expression of LIG4 is shorter.2.The high expression of LIG4 may be involved in the resistance of triple negative breast cancer to paclitaxel.The use of LIG4 inhibitor（SCR7）can enhance paclitaxel-induced apoptosis and reduce the resistance of MDA-MB-231/PTX cells to paclitaxel.3.The LIG4 inhibitor（SCR7）may inhibit the resistance of MDA-MB-231/PTX cells to paclitaxel by inhibiting the non-homologous end joining activity and its alternative pathways（PARP1,XRCC1）.