| BackgroundBreast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in females worldwide, accounting for23%(1.38million) of the total new cancer cases in2008, ranks second only to lung and it is a serious threat to female’s health. Over the past20years, China’s urban female breast cancer incidences was significantly increasing, especially in big cities like Beijing, Tianjin and shanghai. Compared with Europe and other Western countries, the younger patients with breast cancer in our country, the rising incidence of30years old, the peak age of onset in40-49years,10-15years earlier than in Western countries, the harm to people and society more large. Despite the significant improvement in both diagnostic and therapeutic modalities for breast cancer patients, metastasis still represents as the major cause of mortality. Nearly90%of patients with breast cancer deaths are caused by cancer metastasisBreast cancer metastases are regulated by many signaling pathways and the regulation is a complicated period including interactions of many proteins and factors relative to migration. Although many studies have focused on breast cancer metastasis, but the mechanism of metastasis is still not very clear. Therefore, exploring the molecular mechanisms of breast cancer metastasis deeply would contribute to finding new effective therapeutic methods. It is helpful for increasing the survive rate of breast cancer patients.Calcium, a ubiquitous second messenger involved in tumorigenesis, invasion, metastasis and a series of processes. Store-operated calcium channel (SOC), is located on the cell membrane. It is the main channel regulating the Ca2+influx in non-excitable cells. The channel activation is aroused by the Ca2+store depletion. The activation of phosphorylation cascades by either G-protein-coupled receptors or immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors located in the plasma membrane triggers the activation of phospholipase C (PLC) β or γ, respectively, which in turn catalyzes the synthesis of inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) from phosphatidylinositol-4,5-bisphosphate (PIP2). The increase of IP3in the cytoplasm activates specific Ca2+channels located in the ER membranes, referred to as IP3receptors (IP3R), leading to a release of Ca2+stored in the ER into the cytoplasm.Stromal interacting molecule (STIM)1acts as a sensor for the level of Ca2+stored in the endoplasmic reticulum. When the Ca2+stored in the ER is depleted, then activates the extracellular Ca2+influx across the plasma membrane by store-operated calcium entry (SOCE) mechanisms. Orail and STIM1are critical for breast tumor cell migration and metastasis in mice. In highly metastasis of breast cancer cell MDAMB-231using RNA interference downgraded Orail and STIM1or used Ca2+channel pharmacology inhibitor treatment can reduce the animal model of tumor metastasis, and overexpression of STIM1or ORAI1can promote breast cancer cell invasion ability.STIM2could act as a regulator that stabilizes basal cytosolic and ER Ca2+levels. Initial studies reported that knockdown of STIM1strongly reduced SOCE in human Jurkat T and HeLa cells, while overexpression or downregulation of STIM2had a minor or no effect, The function of STIM2in SOCE is still controversial. STIM2is a powerful SOC inhibitor when expressed in HEK293, PC12, A7r5, and Jurkat T cells. STIM1and STIM2had the opposite function, and can coordinate with each other in SOCE signaling pathways.This study focused on the STIM1and STIM2influence on breast cancer cell migration, and the regulatory mechanism of STIM1and STIM2influence breast cancer cell migration, help to elucidate the molecular mechanism of regulating the migration of breast cancer cells provides a new experimental basis, and provides a new clue for transfer of breast cancer treatment targets.ObjectivesTo observed the mRNA expression of STIM1and STIM2in clinical breast tissue To observed The Protein levels of STIM1and STIM2in breast cancer cell lines, To observed the effect of STIM1and STIM2on breast cancer cell migration; To observed RNA silence and over-expression of STIM1on breast cancer cell migration and inhibition, RNA silence and over-expression of STIM2on breast cancer cell migration, to explore the Calcium signal mechanism.Methods1. Extracted and compared STIM1and STIM2mRNA expression in Oncomine database.2. Breast cancer cell lines using RPMI-1640medium containing10%fetal bovine serum in5%CO2%,37℃incubator, passaged2-3times per week, digested with0.25%trypsin, used logarithmic growth cells for the experiment.3. Transwell is used to compare breast cancer cell lines migration ability, Using the method of q-PCR measure STIM1and STIM2mRNA expression in breast cancer cell lines, Western-blot was used to measure the STIM1, STIM2protein expression in breast cancer cell lines.4. Cells were treated by0-50μM G418, MMT assay was used to measure the viability of human breast cancer cell after0-50μM G418treatment, Transwell was used to compare breast cancer cell lines migration ability.Lipofectamine2000was used to transfected siSTIMl and siSTIM2to cells and Western-blot was used to measure the effect of RNA inference. MMT assay was used to measure the viability of human breast cancer cell after transfection. Wound-healing assay and Transwell were used to observing cell migration after transfection. Lipofectamine2000was used to transfected STIM1plasmid and STIM2plasmid and Western-blot was used to measure the effect of overexpression inference. Transwell were used to observing cell migraton after overexpression.5. Cells were treated by0-50μM G418, laser scanning confocal microscope was used to observe Cytoplasmic calcium. Lipofectamine2000was used to transfected siSTIMl and siSTIM2to cells, laser scanning confocal microscope was used to observe Cytoplasmic calcium. Lipofectamine2000was used to transfected STIM1plasmid and STM2plasmid and Western-blot was used to measure the effect of overexpression inference, laser scanning confocal microscope was used to observe Cytoplasmic calcium.6. Cells were treated by0-50μM G418and laser scanning confocal microscope was used to observe the effect on TG-induced SOCE of human breast cancer MDA-MB-231cells. Lipofectamine2000was used to transfected siSTIMl and siSTIM2to cells, laser scanning confocal microscope was used to observe the effect on TG-induced SOCE of human breast cancer MDA-MB-231cells. Lipofectamine2000was used to transfected STIM1plasmid and STIM2plasmid, laser scanning confocal microscope was used to observe the effect on TG-induced SOCE of human breast cancer MDA-MB-231cells.Results1. Compared with normal tissues, the higher expression of STIM1in invasive ductal breast carcinoma, ovarian carcinoma, lung adenocarcinoma, clear renal cell carcinoma, hepatocellular cancer and prostate carcinoma. Compared with normal breast tissues, The lower expression of STIM2in intraductal cribriform Breast adenocarcinoma, invasive ductal breast carcinoma, invasive breast carcinoma, mixed lobular and ductal breast carcinoma, invasive ductal and lobular carcinoma and invasive lobular breast carcinoma.2. Compared with MDA-MB-231, BT549, ZR-75-30Cell lines, MCF-7, T-47D, BT-474Cell lines had lower migration ability. Compared with lower migration cell lines, the mRNA expression of STIM1is higher in high migration group. Compared with higher migration cell lines, STIM2mRNA expression is lower in low migration group. Compared with lower migration cell lines, the protein expression of STIM1is higher in high migration group. Compared with higher migration cell lines, STIM2protein expression is lower in low migration group.3. Transwell is used to compare different breast cancer cell lines migration, q-PCR is used to measure the STIM1and STIM2mRNA levels in breast cancer cell lines,western blot is used to measure the STIM1and STIM2Protein in breast cancer cell lines.4. Compared with control, the viability of human breast cancer MDA-MB-231cells was not decreased after0-50μM G418treatment for24h and48h. Compared with control, the number of migrated MDA-MB-231cells was increase after0-50μM G418treatment. Compared with control, the expressions of STIM1and STIM2protein of human breast cancer MDA-MB-231cells was decreased after siRNA transfection for48h. Compared with control, the viability of human breast cancer MDA-MB-231cells was not decreased after siRNA transfection for24h, Compared with control, the number of migrated MDA-MB-231cells was decreased after siSTIMl transfection for48h. Compared with control, the number of migrated MDA-MB-231cells was increase after siSTIM2transfection for48h.5. Compared with control, maximal Ca2+release elevation (the first peak phase) were not decreased and maximal Ca2+entry elevation (the second peak phase) were increased significantly by0-50μM G418treatment; Compared with control, maximal Ca2+release elevation (the first peak phase) were not decreased and maximal Ca2+entry elevation (the second peak phase) induced by TG were decreased significantly after siRNA-STIM1transfection for48h (P<0.01). Compared with control, maximal Ca2+release elevation (the first peak phase) were not decreased and maximal Ca2+entry elevation (the second peak phase) induced by TG were increased significantly after siRNA-STIM2transfection for48h (P<0.01). Compared with control, maximal Ca2+release elevation (the first peak phase) were not decreased and maximal Ca2+entry elevation (the second peak phase) induced by TG were increased significantly after STIM1Plasmid transfection for48h (P<0.05). Compared with control, maximal Ca2+release elevation (the first peak phase) were not decreased and maximal Ca2+entry elevation (the second peak phase) induced by TG were decreased significantly after STIM2Plasmid transfection for48h (P<0.05).Conclusion1. The mRNA expression of STIM1and STIM2in normal tissues and cancer tissues are difference.2. The mRNA expression of STIM1and STIM2in breast cancer cell lines are difference. 3. Downregulation of STIM1inhibit breast cancer cell migration, overexpression of STIM1promote breast cancer cell migration,and downregulation of STIM2promote breast cancer cell migration, overexpression of STIM2inhibit breast cancer cell migration.4. STIM1and STIM2on breast cancer cell migration regulation is modulated by the inflow of calcium. |
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