STIM1 Knockout Breast Cancer MDA-MB-231 Cells-secreted Exosomal MiR-145 Suppressed Angiogenesis By Targeting IRS-1

Objective:In this paper,we investigated the anti-tumor angiogenesis mechanism of exosomes from STIM1 knockout MDA-MB-231 cells,to provide a new theoretical basis and experimental basis for the clinical diagnosis and treatment of breast cancer.Methods:1.MTT assay was used to determine the anti-proliferative activity of calcium inophore A23187 and SKF96365,inhibitor of Store Operated Calcium Entry (SOCE)on MDA-MB-231 and HUVEC.2.Flow cytometry was used to analyze the effect of A23187 and SKF96365 on the cell cycle and apoptosis of MDA-MB-231 cells.3.Wound healing assay was used to determine the effect of A23187 on MDA-MB-231 cell migration and A23187 treated MDA-MB-231 cell-derived exsomes on HUVEC migration.4.Tube formation assay was used to determine the tumor angiogenesis effect of A23187 and SKF96365 treated MDA-MB-231 cell-derived exosomes.5.Fluorescence quantitative PCR was used to detect the level of angiogenesis related miRNAs in MDA-MB-231 cells,exosomes and exosomes treated HUVEC.6.CRISPR/Cas9 technology was used to establish STIM1 knockout MDA-MB-231 cell lines.7.Western blot was used to investigate the effects of SKF96365 treated MDA-MB-231 secreted exosomes and STIM1-deficient MDA-MB-231 derived exosomes on IRS-1 expression and phosphorylation levels of related pathway proteins,such as c-raf,ERK,p38,Akt,and mTOR.8.The effect of A23187,SKF96365 and STIM1 deletion on calcium ion influx in MDA-MB-231 cell were detected by fluorescence quantitative kit.Results:1.In human triple-negative breast cancer,MDA-MB-231 cell-derived exosomes promote HUVEC proliferation and angiogenesis in a dose-dependent manner,but have no accelerative effect on the proliferation of breast cancer cells themselves.2.A23187 increases the intracellular calcium level of MDA-MB-231,increases secreted exosome content,promotes the angiogenesis and migration of HUVEC cells,and promotes the opening of endothelial barrier.3.SKF96365 reduces the intracellular calcium level of MDA-MB-231,which secreted exosomes inhibit the angiogenesis and migration of HUVEC,and inhibits the opening of the endothelial barrier.4.The Level of miR-145 was significantly up-regulated in SKF96365 treated MDA-MB-231 cells,the secreted exosomes and the exosomes treated HUVEC cells.In addition,the expression of IRS-1 and the phosphorylation levels of related pathway proteins,such as c-Raf,ERK,p38,Akt,and mTOR were also significantly down-regulated in HUVEC treated with exosome from SKF96365 treated MDA-MB-231 cells.5.STIM1 knockout MDA-MB-231 cells-secreted exosomal miR-145 inhibits HUVEC angiogenesis,IRS-1 expression and related pathway proteins,such as c-Raf,ERK,p38,Akt and mTOR phosphorylation levels.6.miR-145 antagomir was used to verify the effect of STIM1 knockout MDA-MB-231 cell-derived exosomal miR-145 inhibition of HUVEC vascularization by targeting ISR-1.Conclusions:1.MDA-MB-231 cell-derived exosomes when intracellular calcium ions increased promoted tumor angiogenesis,whereas the exosomes released when intracellular calcium ions decreased inhibited tumor angiogenesis.2.STIM1 deletion MDA-MB-231 cells-derived exosomal miR-145 inhibited HUVEC angiogenesis by targeting IRS-1 in vitro.