Mechanism Of G2 Phase Cell Arrest In Breast Cancer Cells Induced By NDRG2 Gene Overexpression

At present,the incidence of breast cancer has ranked the first among female malignant tumors.The treatment methods of breast cancer are increasingly sufficient,including surgical treatment,chemotherapy,targeted therapy,endocrine therapy and radiation therapy.Whereas,there are many patients die of breast cancer on account of recurrence and metastasis,which may be due to the resistance to anti-cancer drugs.The need for novel breast cancer treatment targets is becoming more urgent.NDRG2 gene is one of the members of the NDRG gene family.In breast cancer,NDRG2 gene is capable of inhibiting tumor proliferation,Invasion,metastasis ability and accelerate apoptosis progress of cancer cells.This illustrates that NDRG2 gene is a potential anticancer therapeutic target.Previous study has reported that knockdown of NDRG2 gene can suppress breast cancer cell apoptosis.We hypothesized that NDRG2 gene overexpression can induce apoptosis in breast cancer cells,and one of the causes of apoptosis may be cell cycle arrest.First,we detected the effect of NDRG2 overexpression on cell cycle in breast cancer cells,and G2 phase cell cycle arrest was observed.By screening some signaling pathways that can cause G2 arrest,we found that NDRG2 overexpression had the most significant effect on m TOR/p70S6 K signaling pathway.Therefore,this signaling pathway was selected as the research object.This study investigated the mechanism of G2 phase cell arrest in breast cancer cells induced by NDRG2 gene overexpression,in order to illustrate the molecular mechanism of NDRG2 promoting apoptosis.ObjectiveTo explore mechanism of G2 phase cell arrest in breast cancer cells induced by NDRG2 gene overexpression.Methods1.Construct NDRG2 overexpression vector using pc DNA4-his Max C plasmid,and transfect into human mammary gland epithelial cell line,MCF-10 A,and human breast cancer cell lines,MCF-7,SK-BR-3 and MDA-MB-231.2.Use Western blot and RT-PCR to detect the expression level of NDRG2 in the cells transfected with the overexpression plasmid,verify the overexpression of NDRG2.3.Then,use flow cytometry to detect the cell cycle of NDRG2 overexpression cells.4.The key signal molecules in the m TOR/p70S6 K signaling pathway,p-m TOR,p-p70S6 k and p-c-Jun,p-NF-k B were detected by Western blot.5.The m TOR/p70S6 K signaling pathway and c-Jun downstream target NF-k B,AP-1,YY-1 and Cyclin D1 were detected by RT-PCR and Luciferase assay.Results1.Cell cycle analysis result indicated that overexpression of NDRG2 gene is able to shorten the S-phase and prolong the G2-phase of breast cancer cells,induced G2 phase cell cycle arrest.2.Western blot results illustrated that NDRG2 gene overexpression suppresses expressions of p-m TOR,p-p70S6 K,p-NF-k B and p-c-Jun in breast cancer cell lines.3.Luciferase assay results showed that the expression of NF-k B and AP-1 in the NDRG2 overexpression group was significantly inhibited in breast cancer cell lines.4.According to RT-PCR results,overexpression of NDRG2 can significantly reduce the transcriptional level of Cyclin D1 and YY-1.ConclusionsOverexpression of NDRG2 gene is capable of inhibiting m TOR/p70S6 K signaling pathway and c-Jun expression,decrease the expression of downstream targets NF-k B,AP-1,YY-1 and Cyclin D1,and induces G2 phase cell cycle arrest in breast cancer cells.