CDCA4 Regulates Cell Proliferation And Apoptosis In The MCF-7/ADM Human Breast Cancer Cell Line

Background: Breast cancer is a global health challenge.Patients with early-stage breast cancer following treatment with surgery followed by postoperative radiotherapy have a satisfactory survival rate.However,a substantial number of patients with terminal breast cancer are unable to undergo surgery due to symptomatic metastasis.Chemotherapy and other treatments can prolong the survival time of patients with terminal breast cancer.Chemotherapy is widely used for breast cancer treatment,But frequent resistance phenomena tends to limit its effectiveness.The tolerance of these patients is often too weak to accept chemotherapy.In recent year,Gene-targeted therapy has been shown to be a potential effective supplement for conventional therapy for cancer.Multiple types of research determined that knockdown some Drug-resistance-associated genes can preventing even overcoming chemo-resistance.Prophase research has suggested that cell division cycle-associated protein 4(CDCA4)is a downstream gene of the nuclear factor erthyroid 2-related factor 2(Nrf2)signaling pathway that has been shown to regulate the resistance of cancer cells to chemotherapeutic drugs.In addition,the function of CDCA4 in human breast cancer has not been elucidated.Considered together,undertaking further study to investigate the relation between CDCA4 and breast cancer is absolutely necessary.Objective: This study aimed to investigate the association between cell division cycle-associated protein 4(CDCA4)expression level and the proliferation and apoptosis of the Adriamycin-resistant MCF-7/ADM human breast cancer cell line and Further expounded the functions of CDCA4 in breast cancer.This study can finally provide a scientific rationale for potential gene-targeted therapy of breast cancer.Methods: In this study,Adriamycin-resistant breast cancer MCF-7/ADM cells were selected as the model for experiments,which were stably transfected CDCA4-shRNA to silence CDCA4 expression.CDCA4-ShRNA transfection efficiencies were analyzed by fluorescent microscopy.The mRNA and protein level of CDCA4 in MCF-7/ADM cells after transfected CDCA4-shRNA were examined using RT-qPCR and Western blot.MTT assay,colony formation assay and Flow cytometry assay were used to explore the tumor biological behavior of MCF-7/ADM cells,which including proliferation,apoptosis and cell cycle.Results: 1.RT-qPCR and Western blot: CDCA4 shRNA-mediated suppression of expression in the MCF-7/ADM cell line was examined using RT-q PCR analyses.The CDCA4 mRNA expression level of CDCA4 knockdown(KD)group was obviously lower than the negative control(NC)group.Additionally,the rate of KD groups knockout was 58.1%(p <0.05).CDCA4 protein level in the MCF-7/ADM cells was detected using Western blot assay.Western blot results show that CDCA4 protein level of KD group was significantly lower than the NC group(p <0.05).2.Colony formation assay: A colony formation assay was used to evaluate the growth of the breast cancer cells in which CDCA4 was silenced.CDCA4 knockdown in the MCF-7/ADM cells led to the formation of significantly fewer colonies on soft agar,compared with the shCtrl group of cells(P<0.05).3.MTT assay: To further examine the negative effect of CDCA4 knockdown on breast cancer cell growth,an MTT assay was performed.As shown by the results,the proliferation of shCDCA4-transfected group cells was slower,compared with that of the shCtrl transfected-group of cells during the first 5 days following plating of the cells(P<0.05).4.Apoptosis assay using flow cytometric analysis: To investigate whether CDCA4 shRNA transfected cells and shCtrl transfected cells showed differences in apoptotic rate,an apoptosis assay was performed on the experimental and control groups of cells using flow cytometry.We found that percent of cell apoptosis in the KD group,transfected by CDCA4-shRNA,was increased significantly compared with the NC group(P<0.05).5.Flow cytometric analysis of cell cycle: Flow cytometry was used to examine whether the promoting effect of CDCA4 on MCF 7/ADM cell growth and proliferation was mediated through cell cycle progression.In the shCDCA4 transfected group of cells,40.10% of cells were in the G0/G1 phase,29.34% were in the S phase and 30.56% were in the G2/M phase of the cell cycle,which were significantly lower,compared with the percentages in the control group of cells.Conclusion: The expression of CDCA4 was found to be associated with the fate of breast cancer cells.Using RNA interference technology,the results of in vitro experiments indicated that a downregulation in the expression of CDCA4 inhibited proliferation and induced apoptosis inthe MCF?7/ADM human breast cancer cell line.These data provide a scientific rationale for potential gene?targeted therapy of breast cancer.Other potential functions of CDCA4 in breast cancer require further elucidation through additional relevant investigations.