The Role Of STIM2-mediated ROCE In Cell Cycle Regulation Of Breast Cancer Cells

BackgroundBreast cancer ranks first among the world’s female malignant tumors,and its mortality rate is second only to lung cancer.Tumor related death caused by tumor metastasis is the most important factor in the death of breast cancer patients.However,the molecular mechanism of breast cancer metastasis has not yet been clearly understood,and the clinical treatment of breast cancer still lacks effective targets.As a type of malignant tumor,the development of breast cancer is often accompanied by uncontrollable malignant proliferation of cancer cells,which involves a complete cell cycle expression.Stromal interaction molecule 2(STIM2)is a calcium homeostasis sensing receptor that regulates intracellular calcium homeostasis.Cellular calcium signals participate in many cellular biological processes,such as cell proliferation,cell cycle,and epithelial mesenchymal transition(EMT).Receptor-operated Ca2+entry(ROCE)is one kind of calcium signals that regulating calcium influx independent on the state of calcium store.ROCE is risen by activating of receptors on the membrane that leads to extracellular calcium influx.Current reports suggest that STIM2 is involved in SOCE.However,there is no specific report on the relationship between STIM2 and ROCE,and whether STIM2 regulates the proliferation and cycle of breast cancer cells via ROCE is also lack of evidence.ObjectiveWe aimed to explore the relationship between STIM2 and cell proliferation and cell cycle in breast cancer cells,and whether STIM2 can regulate cell proliferation and cell cycle expression through ROCE.Methods1.The lentivirus transfection method was used to construct stable expressing STIM1 and STIM2 breast cancer cell lines and Western blot was used to detect the over-expression and down-regulation of STIM1 and STIM2.2.CCK8 assay,BrdU incorporation assay and plate colony formation assay were used to observe the relationship between STIM2 and cell proliferation.3.Flow cytometry and Western blot were used to detect the relationship between STIM2 and cell cycle.4.Laser confocal microscopy was used to detect the relationship between STIM2 and ROCE.5.Cells were treated with inhibitor SKF-96365 and laser confocal microscopy was used to detect the effect of inhibition of ROCE.6.The effect of ROCE on proliferation of breast cancer cells were detected by CCK8 assay,BrdU incorporation assay and plate colony formation assay.7.Flow cytometry and Western blot were used to detect the effect of ROCE on cell cycle.Results1.Western blot results showed that it was successful using lentivirus transfection to construct stable overexpression and downregulation of STIM1 and STIM2 cell lines.2.Results of CCK8 assay,BrdU incorporation assay and plate colony formation assay showed that downregulation of STIM2 suppresses cell proliferation.3.Flow cytometry and Western blot results indicated that downregulation of STIM2 arrests cell cycle from G1 to S phase via decreasing Cyclin D1 expression.4.Laser confocal microscopy results showed that STIM2 mediates G protein coupling receptor-activated ROCE of breast cancer cells.5.Laser confocal microscopy results showed that SKF-96365 could effectively inhibit ROCE while SOCE is not affected.6.CCK8,BrdU incorporation assay and plate clone formation assay results showed that cells proliferation is suppressed followed by inhibiting ROCE.7.Results of flow cytometry and Western blot showed that inhibition of ROCE arrests cell cycle from G1 to S phase via decreasing Cyclin D1 expression.ConclusionsIn breast cancer cells,STIM2 mediates the G protein coupled receptor(GPCR)-activated ROCE and blocking of ROCE induced G1 phase cell cycle arrest leading to significant decrease in cell proliferation.