| BackgroundLung cancer is the most common malignant tumor in China. Non-small cell lung cancer (NSCLC) is the majority of lung cancer, approximately80%of total malignancies. Multiple genetic and epigenetic changes are involved in the development and progression of NSCLC. Calcium, as the second messenger, is essential signal transduction element involved in cell growth including cell cycle, differentiation, proliferation and apoptosis. The intracellular calcium concentration plays an important role in cell activities, regulated by release from endoplasmic reticulum stores or influx through a variety of Ca2+ion channels. Voltage-gated (VGCC), receptor-gated (ROCC) and store-operated (SOCC) channels in the membrane, along with ryanodine receptors (RynR) and inositol triphosphate receptors (IP3R) at the endoplasmic reticulum (ER) store, provides fluxes of Ca2+to the cytoplasm. Reducing of the Ca2+in ER can result from Ca2+-influx from the extracellular space. SOCC in the membrane is activated by the emptying of the intracelluar Ca2+-stores causing Ca2+influx. This process is name by store-operated calcium entry (SOCE). SOCE plays a vital role in the cell function including emiocytosis, enzyme activity, cell cycle and apoptosis. Now, SOCE induced by SOCC is mostly investigated in the malignant tumor. The most popular channel in SOCE is calcium-release activated calcium (CRAC) channel. STIM1as the ER Ca2+sensor, the highly Ca2+-selective CRAC channel ORAI1as the effector of membrane, expressed in cells. STIM1and ORAI1are essential for tumor cell migration and proliferation in vitro and vivo. But there are few researches about SOCE in lung cancer.ObjectiveWe first investigate STIM1and ORAI1mRNA expression in NSCLC tumor samples. Second, to investigate SOCE of human lung adenocarcinoma A549cell in proliferation, migration and invasion. Then, to investigate the role of STIM1or ORAI1in A549cell in proliferation, migration and invasion. Last, to analyze the correlation between STIM1or ORAI1level and disease-free survival of IIIA NSCLC.Patients and methods1.24NSCLC tumor samples from2007-2009in Cardiothoriac departement, the First Affiliated Hospital of Guangzhou Medical College, were performed in this research. We performed total RNA isolation with Trizol, then cDNA by reverse transcription, STIM1and ORAI1mRNA using Real-time Quantitative PCR.2. A549human lung adenocarcinomal and16-HBE human bronchial epithelial cells was provided by the Cell Bank (Chinese Academy of Sciences, Shanghai, China). Cells were exposed to SKF-96365and NiC12alone. Cells were treated with indicated drugs and incubated for24-72hours at37℃. WST-1(Roche, Basel, CH) was added to each well and incubated for4hours at37℃. The plates were then analyzed on a microplate reader at450nm to determine the absorbance of the samples. The tumor cell migration activity was assayed in Transwell(?) cell-culture chambers. Migration (%) is the ratio of the migrated cells of the lower surface to the total cells in the surface of the filter. For cell invasion assays, only the cells of the lower surface were counted after removing the cells and coated matrigel on the upper surface of the filters. Intracellular [Ca2+] measurements were performed by fura2-AM. Datas were collected and analyzed with InCyte software.3.We silenced STIM1and0RAI1gene expression by siRNA using Nucleofector method. Then we investigated the proliferation, migration and invasion in A549cells as described above.4. We analyzed STIM1or0RAI1level using S-P immunohistochemistry in ⅢA NSCLC.Results1. STIM1/ORAI1mRNA expressionin in NSCLC tumor samplesThe median mRNA of STIM1was0.84(0.19-3.4),1.09±0.82(X±S) in24tumor samples, compared with normal lung tissue,0.83(0.33-2.6),0.91±0.51(P>0.05). The median mRNA of ORAI1was0.95(0.31-4.9),1.51±1.46in17tumor samples compared with normal lung tissue,0.76(0.31-4.0),1.0±0.92(P>0.05). But we found there was significant difference between Orai expression in NSCLC with poor and good differentiation (P=0.028<0.05). And STIM1expression seems higher in adecnocarcinoma than in squaraous carcinoma (P=0.055)2. The role of SOCE in A549cell in proliferation, migration and invasion2.1SOCE in A549and16HBEAfter perfused with Ca2+-PSS solution, different SKF-96365and NiCl2could reduce calcium influx and dose-dependent. We select8-14cells, compared with untreated A549cells (peak Δ[Ca2+]i,482±36nM), cells perfused with SKF-96365had significant decrease in peak Δ[Ca2+]i at1μM (372±17nM),10μM (268±26nM) and50μM (188±23nM)(P<0.05), with NiCl2at50μM (268±33nM),200μM (250±55nM) and500uM (208±47nM) compared with untreated A549cells (P<0.05). Compared with untreated16HBE cells (peak Δ[Ca2+]i,290±53nM), cells perfused with SKF-96365had significant increase in peak Δ[Ca2+]i at lμM (743±93nM) and l0μM (510±32nM)(P<0.001), and significant decrease in peak Δ[Ca2+]i at50μM (180±29nM). Compared with control16HBE cells (peak Δ[Ca2+]i,460±45nM), cells perfused with NiCl2had significant decrease at200μM (337±37nM) and500μM (173±28nM),1000uM(58±6.1nM).2.2Cell proliferation after treated with SOCE inhibitorThe effect of SKF-96365or NiCl2on the proliferation of A549cells was evaluated via WST assay. Compared with the untreated cells, A549or16HBE cells treated with SKF-96365lOuM or NiCl2500uM showed a strong reduction in cell proliferation after24,48,72h exposure (P=0.001). The relative inhibitory rate of SKF-96365on A549and16HBE cells after48hours was51.7%,38%respectively (P <0.05). And the relative inhibition rate of NiCl2on A549and16HBE cells after48hours was49.7%,42.5%respectively.2.3. Cell migration and invasion after SOCE inhibitorIn the preliminary experiment, we found only A649cell had migration and invasion abilities. Different concentration of SKF-96365or NiCl2treatment of A549cells resulted in a significant inhibition of migration and invasion compared to untreated cells. The migration rate of lOuM SKF-96365treatment or500uM NiCl2of A549cells was10%±2%,19%±4.3%(mean±SD) respectively. Compared with untreated cells (49±3.1%), there was a significant decrease in migration (P<0.01), even in the low concentration of SKF-96365(luM,20%±2.6%) or NiCl2(50uM,32%±5.6%)(P<0.05). And the similar results were seen in the invasion assay, the migrated cells of SKF-96365lOuM or500uM NiCl2treating were110±26,320±52respectively compared with control A549cells (500±45) in one field (P<0.05) too. Also the low concentration of SKF-963651uM could reduce the invasion of tumor cells (200±36) significantly (P<0.05).3.The role of STIM1or ORAI1in A549cell in proliferation, migration and invasion3.1Cell proliferation after silencing STIM1or0RAI1gene expressionWe found there is no change in A549cell proliferation after silencing STIM1using siRNA. The cell inhibition (%) was0.04±0.015,0.05±0.01and0.07±0.01after24,48,72hours. There is no change in A549cell proliferation after silencing ORAI1using siRNA. The cell inhibition (%) was0.06±0.01,0.14±0.01and0.06±0.01.3.2Cell migration and invasion after silencing STIM1or ORAI1gene expressionWe found there is a significant difference in cell migration between negative interefere group and STIM1interfere group. Compared with negative group (migration rate,0.38±0.02), the migration of STIM1group is significantly decreased (0.14±0.04)(P=0.001<0.01). The migrated cells in invasion assessment in STIM1group are significantly decreased (246±23) compared with STIM1group (80±11)(P=0.000<0.01).There is a significant difference in cell migration between negative interefere group and ORAI1interfere group. Compared with negative group(migration rate,0.17±0.02), the migration of ORAI1group is significantly decreased (0.08±0.01)(P=0.003<0.01). The migrated cells in invasion assessment in ORAI1group are significantly decreased (176±21) compared with ORAI1group (82±8)(P=0.002<0.01)4. The correlation between STIM1or ORAI1level and disease-free survival in stage ⅢA NSCLC4.1. Patients characteristics and STIM1or ORAI1expression STIM1or ORAI1positive was visible in the tumor cell plasma or membrane.27samples expressed low level of STIM1in male (64.3%, P=0.055), and STIM1low level was observed in15squamous cell carcinoma (71.4%, P=0.084). Low ORAI1level was detected in18squamous cell carcinoma (85.7%,P=0.067)4.2. Clinical outcome of patients with STIM1low and high levelMale patients in stage ⅢA with STIM1low level had better disease-free survival than those with STIM1high level (21months vs12months, P=0.06). And in squamous cell carcinoma patients, STIM1low level predicted better survival too compared with high level (33months vs9months, P=0.07)Conclusions1. STIM1and ORAI1mRNA could be detected in NSCLC tumor tissues. And STIM1expression may be higher in adenocarcinoma, poor differentiation NSCLC tissues express ORAI1gene more higher.2. We found SOCE existed in A549and16HBE cells. SOCE play a partial role in the proliferation of these cells. SOCE plays an important role in cell migration and invasion in A549cells.3. We found STIM1or ORAIl expression had no influence on the proliferation of A549cell, but in vitro they play an important role in the cell migration and invasion.4. In squamous cell carcinoma or male NSCLC patients with stage ⅢA,low level STIM1expression might pedict better disease-free survival. |
PDF Full Text Request