Mechanism Of TOX3 Gene-mediated Apoptosis Of Breast Cancer Celland The Intervention Effect Of DP

Objectives:To investigate the relationship of the expression of TOX3 and the development and clinical pathology of breast cancer.Reveal the effect of TOX3 on the cell apoptosis in breast cancer and its influence on mitochondria apoptosis pathway by regulating the intracellular expression of TOX3.Discuss the effects of DP on breast cancer cell apoptosis,expression of TOX3 and mitochondria apoptosis pathway.Investigate further the role of TOX3 in the apoptosis of breast cell induced by DP.Methods:Imunohistochemstry was used to detect the expression of TOX3 in breast cancer tissues and normal breast tissues.RNA interference was performed to silence the TOX3 expression to construct the breast cell line with TOX3 gene stably silenced.Lentiviral expression vectors were used to construct the cell lines with stable overexpression TOX3 gene.MTT and plate clone formation assay were used to measure cell proliferation.Flow cytometry was performed to detect the rate of early apoptosis,late apoptosis and cell cycle.Real-time PCR and Western blot were used to measure the mRNA and protein expression of corresponding gene in breast cancer tissues and cells.Results1.The expression of TOX3 was found much higher in breast cancer tissue than that in normal breast tissue and its expression increased obviously in T3,T4 stage and the tissue with lymph node metastasis.2.TOX3 protein level was high in ZR-75-1 cells but low in MDA-MB-231 cells.3.The sequencing results suggested that the sequence of TOX3 interferential recombinant plasmid and overexpression recombinant plasmid constructed were correct.Stable transfection cell line TOX3-shRNA and MDA-MB-23 1-TOX3 of TOX3 gene was constructed successfully.Real-time PCR and Western blot results showed that the interference efficiency in TOX3-shRNA-3 group was highest,and the level of TOX3 in overexpression cell MDA-MB-231-TOX3 was up-regulated obviously(P<0.05).4.After the silence of TOX3 gene,the ability of cell proliferation and monoclonal formation were deceased(P<0.05),the rate of cells in G0/G1 phase were increased and typical morphological characteristic of apoptosis was observed.Whereas the overexpression of TOX3 enhanced the cell proliferation activity,the number of monoclonal formation and the rate of cells in G2/M phase were increased significantly(P<0.05).After serum starvation for 24h and 48h,the growth inhibition rate was reduced in TOX3 overexpression group,the cells rate of early apoptosis and late apoptosis were reduced(P<0.05)after serum starvation for 48h compared with the negative control group.5.Compared with the negative control group,the mRNA and protein expression of TOX3 and Bcl-2 in TOX3-inter ferenced group was down-regulated,but the mRNA expression of Bax,Caspase-9 and Caspase-3 and the protein expression of Bax,Cyt-c,Cleaved caspase-9 and Cleaved caspase-3 were up-regulated(P<0.05).However,the mRNA and protein of TOX3 and Bcl-2 were increased and Caspase-3 were decreased(P<0.05)in TOX3 overexpression group,but there was no distinctly difference in the expression levels of Bax(P<0.05)compared with the negative control group.6.After the treatment of DP,the prolif’eration of ZR-75-1 cells was repressed in a time and dose dependence manner,the rate of cells in G0/G1 phase and early apoptosis,late apoptosis were increased(P<0.05).The typical morphological characteristic of apoptosis was observed under the transmission electron microscope7.The mRNA and protein expression of TOX3 and Bcl-2 was down-regulated(P<0.05),Bax and Caspase-3 was up-regulated in each treatment group of DP compared with the control group(P<0.05)8.Compared with TOX3-interferenced group and DP group,the cell proliferation activity was reduced and the apoptosis rate was increased in TOX3 interference+DP group significantly(P<0.05).Whereas the mRNA and protein expression of TOX3 and Bcl-2 were further down-regulated(P<0.05),simultaneously the expression level of Bax and Caspase-3 were up-regulated(P<0.05).Conclusions1.TOX3 protein was high expression in breast cancer tissues and related with clinical pathological factors and lymph node metastasis.2.The breast cancer cell line TOX3-shRNA with TOX3 stabled interference and cell line MDA-MB-231-TOX3 of TOX3 gene overexpression were constructed successfully.3,The silence of TOX3 gene inhibited the proliferation of ZR-75-1 cells and induced cell apoptosis,The overexpression of TOX3 promoted MDA-MB-231 proliferation and protected the cells.The machanism may involve in mitochondrial pathway.4.DP repressed the proliferation of ZR-75-1 cells and induced the cell apoptosis.The silence of TOX3 gene strengthened the effect of DP on the apoptosis of ZR-75-1 cell.The mechanism may be both related to TOX3 which involved in the mitochondrial pathway.