Role Of SPZ1 In Regulating Chemoresistance And EMT In Drug Resistant Breast Cancer

Aim:To investigate the effect of SPZ1 on breast cancer EMT phenotype and drug resistance in MCF-7/ADM and MCF-7/PTX cells.Define the target gene regulated by SPZ1,and whether the EMT phenotype and drug resistance of MCF-7/ADM and MCF-7/PTX cells are regulated by the target gene.Methods:1.In this study,sensitive breast cancer cells MCF-7/WT and drug-resistant breast cancer cells MCF-7/ADM and MCF-7/PTX were selected as study subjects.The expression of SPZ1 and Twist1 was reduced in MCF-7/ADM or MCF-7/PTX cells by transfected with small interference RNA?siRNA?.Cell viability was determined using IC₅₀ assay.Wound healing assay was used to detect the cell migration.Transwell chamber was used to detect the cell migration and invasion.The protein expression of SPZ1,Twist1,P-gp,EMT markers including E-cadherin,Vimentin were detected by Western blot.2.The correlation between SPZ1 and Twist1 expression was detected in clinical pre-chemotherapyandpost-chemotherapybreastcancersamplesby immunohistochemistry.3.MCF-7/ADM cells were s.c.injected into the flanks of female nude mice?5×10⁶ cells per mouse?.When the tumors reached¹00 mm³,animals were also injected one time every 3 d at the tumor sites with SPZ1 siRNAs,Twist1 siRNAs or scrambled siRNA as control.Tumor size was measured once or twice a week using a caliper.We used western bolt to detect the changes of SPZ1 and Twist1 in tumor tissues.We used immunofluorescence staining to detect the changes of P-gp in tumor sections.Results:1.The SPZ1 protein in the MCF-7/ADM and MCF.-7/PTX cells has higher expression levels.SPZ1 siRNA treatment increased sensitivity of MCF-7/ADM or MCF-7/PTX cells to ADM or PTX,and inhibited EMT phenotype.We also found that the expression of Twist1 protein was inhibited.Similarly,Twist1 siRNA treatment increased sensitivity of MCF-7/ADM or MCF-7/PTX cells to ADM or PTX,and inhibited EMT phenotype.2.The results of immunohistochemistry showed that SPZ1 and Twist1 levels were higher in clinical post-chemotherapy breast cancer samples,which was consistent with in vitro studies.3.We subcutaneously injected nude mice with MCF-7/ADM cells and the tumor continued to grow in size under ADM treatment.We found that injection of SPZ1siRNAs or Twist1 siRNAs at tumor sites substantially reduced the tumor growth.The knockdown effects of SPZ1 or Twist1 siRNAs were confirmed by SPZ1 and Twist1protein levels.In addition,we found that SPZ1 or Twist1 siRNAs attenuated expression of P-gp by immunofluorescence analysis.Conclusion:SPZ1 plays an important role in regulating drug resistance and EMT phenotype in MCF-7/ADM and MCF-7/PTX cells,which is mediated by Twist1.