Effect Of CDCA4 On Proliferation And Apoptosis Of Triple Negative Breast Cancer Cell Line MDA-MB-231 In Vitro And In Vivo

Background:Breast cancer is the most common malignant tumor to female in worldwide.Triple-negative breast cancer(TNBC;ER-,PR-,HER-2-)is a special molecular subtype of breast cancer with high invasion and high recurrence and metastasis.Due to the lack of effective targets and treatment options,the patient’s survival prognosis is very poor.Therefore,the identification of novel and effective strategies to control TNBC is urgently required.Our previous research evidence suggests that CDCA4 is a downstream gene of the Nrf2-related pathway.CDCA4 and Nrf2 can regulate the proliferation of breast cancer resistant cells MCF-7/ADM in vitro.For drug-insensitive triple-negative breast cells,CDCA4 may have similar regulatory effects.Currently,studies on CDCA4 and triple-negative breast cancer have not been reported.Therefore,this study was to investigate the effect of CDCA4 on the biological function of triple-negative breast cancer cells in vitro,and to study the growth regulation of CDCA4 on breast cancer xenografts in nude mice,and provide new ideas for the treatment of triple-negative breast cancer patients.Objective:To investigate the effect of knockdown of CDCA4 expression on the growth and proliferation of TNBC MDA-MB-231 cells in vitro and in nude mice.To elucidate the initial mechanism(apoptosis and cell cycle)of CDCA4 regulation of MDA-MB-231 cell growth and proliferation,and provide a research basis for the selection of targeted therapy for TNBC.Methods:qRT-PCR was used to detect the difference of CDCA4expression levels in human breast cancer cell lines and normal breast cell line.Short hairpin RNA(shRNA)interference technology was used to knockdown the expression of CDCA4 in TNBC MDA-MB-231 cells.MTT assay and celigo image counting method were used to detect the proliferation of MDA-MB-231cells before and after CDCA4 knockdown.Flow cytometry was used to detect apoptosis and cell cycle distribution.Animal experiments were used to examine the effect of knockdown of CDCA4 on the growth of MDA-MB-231 cell xenograft in the nude mice.Result:(1)RT-PCR results showed that the mRNA expression levels of CDCA4 in three breast cancer cell lines were higher than in a normal mammary gland cell line(P<0.05).Additionally,CDCA4 expression was higher in breast cancer tissues than in normal tissues.(2)Lentiviral transfection with CDCA4-shRNA can effectively knock down the expression level of CDCA4 gene in MDA-MB-231 cells,and the knockdown efficiency is 68.9%(P<0.05).The stable transfection model of MDA-MB-231 cell line was successfully constructed and can be used as a cell model for subsequent experiments.(3)The MTT assay and celigo image counts showed that the number and proliferation of MDA-MB-231 cells in the CDCA4 expression knockdown group(shCDCA4)were significantly lower than those in the negative control group(shCtrl).(4)Flow cytometry showed that the apoptosis rate of MDA-MB-231 cells in the CDCA4 expression knockdown group(shCDCA4)was significantly increased compared with the negative control group(shCtrl)(3.72±0.10%vs9.56±0.53%,shCtrl vs shCDCA4,P<0.05).(5)Flow cytometry assy results showed that the distribution ratio of MDA-MB-231 cells in the CDCA4 knockdown group was changed compared with the negative control group(G0/G1 phase 62.62±0.69%vs 53.05±3.52%;S phase 23.79±2.92%vs 37.67±1.10%;G2/M phase 13.57±2.84%vs 9.29±2.48%;shCtrl vs shCDCA4,P<0.05).Knockdown of CDCA4 expression mainly induces the arrest of the S phase of MDA-MB-231 cell cycle.(6)The results of xenograft experiments in the nude mice showed that the growth ability of xenografts in nude mice of MDA-MB-231 cells in the CDCA4knockdown group(KD)was lower than that in the negative control group(NC)(volume,476.66±400.84mm~3 vs 146.25±204.72mm~3;weight,0.508±0.378g vs0.193±0.280g;NC vs KD,P<0.05).Conclusion:The experimental evidence in this study supports CDCA4 as a tumor-promoting gene and is closely related to the growth regulation of triple-negative breast cancer MDA-MB-231 cells.Using RNA interference technology to knock down the expression of CDCA4 can effectively inhibit the growth and proliferation of MDA-MB-231 cells in vitro and in vivo.In the future,CDCA4 can be a potential target for the treatment of triple-negative breast cancer patients.