Stimulation Of Orai1 Channel By Nucleolin Promotes The Proliferation Of Breast Cancer Cells

Background: Breast cancer is the first malignant tumor that threatens women’s health.The pathogenesis of breast cancer is very complex,and abnormal calcium signal is closely related to the occurrence and development of tumors.Intracellular calcium ion is an important second messenger mediating various cellular functions,and the mechanism involved in Ca2+ homeostasis is the basis of many physiological and pathological processes.The complex Ca2+ signaling system consists of Ca2+ channels,calcium pumps,exchangers,etc,which strictly control the concentration of Ca2+ in the cytoplasm.In tumor cells,the expression or activation state of various calcium ion channels and calcium pumps has changed significantly.Orai1 protein is a very important calcium channel in cells.Orai1-mediated calcium influx is the predominant way for non-excited cells to enter the cell,and is called store-operated Ca2+ entry（SOCE）.Over-expression and activation of Orai1 have been reported in breast cancer.However,its molecular mechanisms are still not very clear.Here,we demonstrated that Nucleolin（NCL）was a novel interacting partner of Orai1.NCL is a multifunctional nucleocytoplasmic protein and is upregulated in human breast tumors.In this study,we aimed to investigate the interaction mechanism between Orai1 and NCL,and its role in the development of breast cancer.Objective: To study the mechanism of Nucleolin（NCL）in tumor proliferation and the new activation mechanism of calcium channel Orai1,so as to provide more theoretical basis for the two anti-tumor targets of NCL and calcium channel Orai1.Methods: Mass spectrometry identification,Western-blot and Co-IP assays were used to detect whether NCL binds to the N-terminus of Orai1;pulldown and immunocolocalization assays were used to detect the main role of NCL and Orai1;immunohistochemical experiments were used to detect NCL in breast cancer tissue and normal breast tissue Western-blot analysis of PCNA expression in MCF-7 cells with high expression of NCL;Western-blot analysis of AKT,P-AKT,PKC and P-PKC protein expressions;MTT,colony formation,EDU and the proliferation of breast cancer cells was detected by trace assay;the calcium ion level in cells was detected by calcium imaging assay and confocal fluorescence calcium assay;the tumor-bearing experiment in nude mice was used to measure the subcutaneous tumor in nude mice by injecting Orai1 polypeptide and calcium channel inhibitor 2-APB.Volume and weight;use analysis of variance and t test（SPSS20.0）for statistical analysis,P<0.05 to judge whether there is statistical significance.Results:Mass spectrometry identification found that NCL binds to the N-terminus of Orai1,and Western-blot proved that NCL could interact with the N-terminus of Orai1.Pulldown and immunocolocalization experiments demonstrated that NCL can interact with Orai1,and the main regions of action are fragments 1-283 and 630-710.Immunohistochemical results showed that NCL was expressed at a higher level in breast cancer cells.Western-blot experiments showed that the PCNA of NCL was significantly enhanced,and the expression levels of P-AKT and P-PKC were increased.MTT experiment,colony formation experiment,EDU experiment,scratch experiment found that NCL can promote the proliferation of breast cancer cells.The results of calcium imaging experiments and confocal fluorescence calcium measurement experiments showed that NCL can promote calcium influx by regulating the calcium channel Orai1.Nude mice bearing tumor experiments found that Orai1 polypeptide and calcium channel inhibitor 2-APB can lead to subcutaneous tumor body and weight loss in nude mice.Conclusion: NCL activates SOCE by interacting with Orai1 at the C-terminus.NCL regulates the activation of the calcium channel Orai1 to promote the proliferation of breast cancer cells.Orai1 inhibitors and blocking interacting polypeptides can effectively exert anti-tumor effects.