The Mechanism Of TGF? Affecting The Proliferation And Apoptosis Of Breast Cancer Cells By Regulating NK1R-Tr

Objectives: In this study,we analyzed the differences in the expressions of TGF? and NK1 RTr in breast cancer tissue and para-cancer tissue samples,and explored the differences in the expressions of TGF? and NK1R-Tr in tissues and their relationship.Subsequently,we used breast cancer cell lines to further study the regulation of intracellular signaling pathways of the two and their effects on cell proliferation,cell cycle and apoptosis,so as to provide new evidence for the prevention and treatment of breast cancer.Methods: 1.50 cases of breast cancer tissue and paracancerous tissue samples from the Department of Breast Oncology,Tianjin Medical University Cancer Hospital were selected.The clinical diagnosis were confirmed by pathological examination.Immunohistochemical staining and Western Blot were used to detect the expression of TGF? and NK1R-Tr in cancer tissues and paracancerous tissues.The relationship between TGF? and NK1R-Tr in cancer tissues and prognosis and the correlation between them were also analyzed.2.In MDA-MB-231 and BT549 breast cancer cell lines,TGF? stimulation was added after the knockdown of NK1 R and the addition of NK1 R antagonist,respectively,and the effects of TGF? and NK1R-Tr on cell growth and proliferation were detected by CCK8 and clonal formation experiments.The influence of TGF? and NK1R-Tr on cell growth cycle was detected by flow cytometry.At the same time,we analyzed the changes of cell distribution in each period.Annexin V/PI staining technique was used to detect the effects of TGF? and NK1R-Tr on the apoptosis of tumor cells.Then we performed Western Blot analysis on the changes of proteins related to the proliferation,cell cycle and apoptosis signaling pathway.With high TGF? expression and decreased NK1 R expression were respectively constructed in MDA-MB-231 cells,which were injected under the skin of nude mice.The nude mice were uniformly killed 32 days later,and the tumor was removed.The tumor size and weight were measured for statistical analysis and comparison of the differences between the groups.3.Two breast cancer cell lines,MDA-MB-231 and BT549,were selected to detect the m RNA and protein levels of NK1R-Tr at different time points after the addition of TGF? stimulation.At the same time,the localization of Smad4,an important molecule in the downstream signaling pathway,was observed by immunofluorescence before and after TGF? stimulation.The regulation of Smad4 on the activity of NK1 R promoter was confirmed by chromatin immunoprecipitation and dual luciferase reporter assay,and the regulation of TGF? on NK1R-Tr was detected after Smad4 knockdown.Results: 1.The expression levels of TGF? and NK1R-Tr in breast cancer tissues were higher than those in adjacent tissues.TGF? and NK1R-Tr expression were positively correlated in human breast cancer tissues.2.NK1 R knocked down or addition of NK1 R inhibitor in MDA-MB-231 and BT549 attenuated the effects of TGF? on cell proliferation and reduced the phosphorylation levels of downstream ERK and AKT.The proportion of breast cancer cells in G2/M phase significantly increased,the expression of cyclin B1 in cell cycle protein decreased,the expression of P21 protein increased,and the effect could be weakened by TGF? addition.The apoptosis of breast cancer cells was significantly increased,the activity of apoptosis-related protein Caspase-3 was increased and the expression of Bcl-2 was decreased.3.In MDA-MB-231 and BT549 cell lines,with the addition of TGF? stimulation,the m RNA and protein expression levels of NK1R-Tr increased gradually with the increase of time.m RNA and protein levels of NK1R-Tr decreased after Smad4 knockdown.After TGF? treatment,the binding capacity of Smad4 residual NK1 R promoter was enhanced and luciferase activity was also increased.After Smad4 knockdown,the activity of NK1 R promoter region was weakened.4.Compared with the control group,both high expression of TGF? and NK1 R promoted tumor growth in mice,and the tumor volume and weight of mice with both high expressions increased significantly.Conclusions: In this study,the expression levels of TGF? and NK1R-Tr in breast cancer tissues were higher,and both correlated with poor prognosis of patients.TGF? regulates the expression of NK1R-Tr by Smad4.At the same time,TGF? promoted cell proliferation and inhibited apoptosis by acting on NK1R-Tr.Therefore,our study mainly investigated the regulation of TGF? on the expression of truncated neurokinin 1 receptor in breast cancer and the effect on the function of breast cancer cells.