钙池操纵性钙内流在人肺腺癌A549细胞中的研究
摘要
研究背景
肺癌是目前发病率和死亡率增长最快、对人类健康和生命威胁最大的恶性肿瘤。肺癌中80%是非小细胞肺癌(non-small cell lung cancer. NSCLC),病理上以鳞癌、腺癌和大细胞癌为主。因此研究肺癌的发生发展机制,寻找有效的治疗靶点,对于改善NSCLC预后,实施个体化治疗尤为重要。钙离子(Ca2+)作为第二信使,在细胞信号传导通路中起着重要的作用,调控细胞周期、分化、增殖和凋亡。近年来关于钙池操纵性钙通道(SOCC)的研究越来越多,由SOCC介导的钙池操纵性钙内流(SOCE)是非兴奋细胞例如上皮细胞产生Ca2+内流的主要方式。研究发现SOCE信号通路中的SITM1及ORAI1蛋白,由于其在介导SOCE中的作用而受到广泛关注。STIM1主要存在于细胞内质网膜上,在钙池消耗后,它作为一种“钙池信号感受蛋白”感受钙池充盈状态,并聚集成"puncta"向细胞膜移动靠近,而非嵌入到细胞膜中,此后STIM1通过羧基端与细胞膜上的SOCC通道蛋白如ORAI1的相互作用而开放钙通道,形成SOCE。STIM1和oRAI1介导的SOCE对上皮细胞的增殖、分化至关重要,因为多数肿瘤来源于上皮细胞,它们在肿瘤细胞中也陆续有相关的研究报道出现。NSCLC多来源于支气管上皮细胞,因此我们选择人肺腺癌细胞A549,研究SOCE在肺癌细胞中的表达情况；研究STIM1/ORAI1介导的SOCE对肺癌细胞的生物学行为如增殖、迁移和侵袭功能的影响；研究NSCLC患者手术肿瘤组织中STIM1或ORAI1表达和生存预后的关系。
研究目的
首先研究人NSCLC肿瘤组织中的SOCC通道STIM1和ORAI1的mRNA表达情况；其次在人肺腺癌细胞系A549中,研究SOCE与细胞增殖、迁移和侵袭的关系；然后研究STIM1和ORAI1蛋白在A549细胞增殖、迁移和侵袭功能中的作用；最后研究NSCLC患者手术肿瘤组织中STIM1或ORAI1蛋白表达和生存预后的关系。
研究方法
1、我们共收集2007-2009年NSCLC术后肿瘤组织和配对远癌正常肺组织(距肿瘤>5cm)24例,标本来自于广州医学院第一附属医院呼吸疾病研究所胸心外科。所有患者术前未行放化疗和其他抗肿瘤治疗。采用Trizol法提取组织总RNA,逆转录成cDNA,荧光定量PCR方法检测组织STIM1和ORAI1mRNA表达。
2、人肺腺癌A549细胞,人支气管上皮细胞16HBE购自中国科学院典型培养物保藏委员会细胞库。细胞增殖实验中,A549细胞和16HBE细胞中分别加入SOCE抑制剂SKF-96365或NiCl2。应用罗氏公司WST-1细胞增殖检测试剂盒检测24、48、72h的OD值,细胞增殖抑制率=1-实验组OD值／正常对照组OD值。A549细胞迁移和侵袭实验是应用Transwell小室法,迁移率=细胞迁移总数-上层未迁移细胞数／细胞总数,侵袭时检测迁移到Transwell小室的下室的细胞数。SOCE细胞钙功能测定是应用Fura-2Am荧光素方法检测,InCytIm2软件收集数据和分析。
3、我们设计合成siRNA, Nucleofector细胞核转染方法瞬时沉默STIMIIORAH基因表达,然后检测A549细胞的增殖、迁移和侵袭功能的改变,余检测方法同上。
4、我们应用S-P免疫组化方法检测70例ⅢA期NSCLC患者手术肿瘤组织STIM1和0RAI1蛋白表达,统计学分析和患者无疾病生存预后的关系。
研究结果
1.NSCLC肿瘤组织中STIM1/ORAI1mRNA的表达
24例患者肿瘤组织STIM1mRNA相对表达量中位值0.84(0.19-3.4),1.09±0.82(X±S)；正常组织STIM1mRNA相对表达量中位值0.83(0.33-2.6)0.91±0.51(P>0.05)。17例患者肿瘤组织ORAI1mRNA相对表达量中位值0.95(0.31-4.9),1.51±1.46；正常肺组织ORAI1mKNA相对表达量中位值0.76(0.31-4.0),1.0±0.92(P>0.05)。在亚组分析中,我们发现腺癌和鳞癌之间的STIM1、ORAI1mKNA的表达无统计学差异(P>0.05),但腺癌的STIM1表达有高于鳞癌的趋势(P=0.055)；低分化肿瘤组织中ORAI1表达要高于分化较好的肿瘤和正常肺组织(P=0.028<0.05),而STIM1却没有类似发现。
2.SOCE对A549肺腺癌细胞的增殖、迁移和侵袭的影响
2.1A549和16HBE细胞的SOCE
在含钙PSS溶液灌流后,不同浓度的SKF-96365和NiCl2都可以减少钙内流,并呈浓度依赖性。我们选取8-14个细胞,相对于未处理过的A549细胞(钙内流峰值Δ[Ca2+]i,482±36nM), SKF-96365处理过的细胞出现△[Ca2+]i下降(P<0.001)：其中SKF-963651μM (372±17nM);10μM (268±26nM);50μM (188±23nM)。相对于未处理的A549细胞(钙内流峰值Δ[Ca2+]i,482±36nM), NiCl2处理过的细胞出现△[Ca2+]i下降(P<0.001)：其中NiCl250μM(268±33nM);200μM (250±55nM);500μM(208±47nM)。
在含钙PSS溶液灌流后,低浓度的SKF-96365增加16HBE细胞钙内流,但高浓度的SKF-96365才减少细胞钙内流。我们选取9-11个细胞,相对于未处理过的16HBE细胞(钙内流峰值Δ[Ca2+]i,290±53nM),低浓度SKF-96365处理过的细胞出现△[Ca2+]i上升(P<0.001)：其中SKF-963651μM (743±93nM);10μM (510±32nM),但高浓度SKF-9636550μM处理后细胞钙内流减少(180±29nM)。相对于未处理过的16HBE细胞(钙内流峰值Δ[Ca2+]i,460±45nM), NiCl2处理过的细胞出现△[Ca2+]i下降(P<0.001)：其中NiCl2200μM (337±37nM);500μM(173±28nM);1000μM(58±6.1nM)。
2.2SOCE抑制剂处理后的A549和16HBE细胞增殖实验
我们首先选择不同浓度SKF-96365处理A549细胞,48h后观察细胞增殖抑制率,结果显示：SKF-963651μM(33%±5%);10μM(46%±4%);50μM(58%±1%),因此我们选择SKF-9636510μM进行下一步的实验。A549细胞经过SKF-9636510μM处理24h、48h和72h,我们发现不同时间点细胞的增殖受到明显的抑制,在24h、48h和72h的抑制率分别为20%±3%,52%±4%,73%±6%。16HBE细胞经过SKF-9636510μM处理24h、48h和72h,发现不同时间点细胞的增殖受到抑制,在24h、48h和72h的抑制率分别为14%±4%,38%±7%；67%±3%。
我们首先选择不同浓度NiCl2处理A549细胞,48h后观察细胞增殖抑制率,结果显示：NiCl2200μM (44%±3%);500μM (50%±4%);1000μM(64%±10%),因此我们选择NiCl2500μM进行下一步的实验。A549细胞经过NiCl2500μM处理24h、48h和72h,我们发现不同时间点细胞的增殖受到抑制,在24h、48h和72h的抑制率分别为12%±4%,50%±4%,62%±9%。16HBE细胞经过NiCl2500μM处理24h、48h和72h,我们发现不同时间点细胞的增殖受到抑制,在24h、48h和72h的抑制率分别为14%±4%,43%±3%,63%±4%。
2.3SOCE抑制剂处理后的A549细胞的迁移和侵袭
我们在预实验中发现A549细胞有迁移能力,而16HBE细胞无迁移能力。我们发现不同浓度的SKF-96365和NiCl2可以明显抑制A549细胞的迁移。相对于未处理过的A549细胞(迁移率,49%±3%),SKF-96365处理后的A549细胞的迁移率降低：SKF-963651μM(20%±3%);10μM(10%±2%);50μM(8%±2%)(P<0.001)。相对于未处理过的A549细胞(迁移率,47%±2%),NiCl250μM (32%±6%);200μM (30%±5%);500μM (19%±4%);1000μM(11%±4%)(P<0.001)。实验中,我们还发现低浓度的SKF-963651μM和NiCl250μM都可以明显抑制A549细胞的迁移。
不同浓度的SKF-96365和NiCl2可以明显抑制A549细胞的侵袭。相对于未处理过的A549细胞(6个视野迁移细胞,503±45个),SKF-96365处理后的A549细胞的迁移细胞减少：SKF-963651μM(210±36个)；10μM(110±26个)；50μM(20±5个)(P<0.001)。相对于未处理过的A549细胞(6个视野迁移细胞,503±45个),NiCl2200μM(396±55个)；500μM(320±53个)；1000μM(100±30个),其中NiCl2500μM和1000μM处理后的迁移细胞相对于未处理组减少(P<0.01)
3. STIM1/ORAI1在A549细胞增殖、迁移和侵袭功能中的作用
3.1siRNA瞬时沉默A549细胞STIM1/ORAI1基因表达
我们应用siRNA沉默A549细胞STIM1基因表达,48h基因瞬时沉默效率最高,可达84%,72h蛋白瞬时沉默效率最高,约70%。我们应用siRNA沉默A549细胞ORAI1基因表达,24h基因瞬时沉默效率最高,可达91%,48h蛋白瞬时沉默效率最高,约80%。
3.2A549细胞STIM1/ORAI1基因表达沉默后的SOCE
我们在瞬时干扰后72h进行钙功能的测定。相对于Negative干扰组(钙内流峰值Δ[Ca2+]i,284±52nM), STIM1干扰组出现了SOCE的下降,约1/3(钙内流峰值Δ[Ca2+]i,194±28nM)(P<0.001)。我们在瞬时干扰后48小时进行钙功能的测定。相对于Negative干扰组(钙内流峰值Δ[Ca2+]i,145±47nM) ORAI1干扰组出现了SOCE的下降,约1/3(钙内流峰值Δ[Ca2+]i,92±24nM)(P<0.05)。
3.3沉默A549细胞STIM1/ORAI1基因表达后的细胞增殖试验
我们发现siRNA瞬时沉默STIM1表达后,A549细胞增殖功能没有明显改变。STIM1或ORAI1siRNA干扰后A549细胞24-96h生长曲线和Negative干扰组无差异。
3.4沉默A549细胞STIM1/ORAI1基因表达后的细胞迁移和侵袭试验
我们发现siRNA瞬时沉默A549细胞STIM1表达后,72h后我们应用Transwell小室法检测细胞迁移率。相对于Negative组(迁移率,38%±2%)STIM1干扰组(迁移率,14%±4%),因此STIM1干扰组的细胞迁移率有下降(P<0.01)。siRNA瞬时沉默A549细胞STIM1表达后,72h后我们应用Transwell小室法检测细胞侵袭。6个视野中,Negative组(迁移细胞,246±23个),STIM1干扰组(迁移细胞,80±11个),因此STIM1干扰组的细胞侵袭实验中迁移细胞有减少(P<0.001)。siRNA瞬时沉默A549细胞ORAll表达后,48h后我们应用Transwell小室法检测细胞迁移率。相对于Negative组(迁移率,30%±12%),ORAI1干扰组(迁移率,12%±5%),与对照组相比ORAIl干扰组的细胞迁移率有下降的趋势,虽然无统计学意义(P=0.084)。siRNA瞬时沉默A549细胞ORAI1表达后,48小时后我们应用Transwell小室法检测细胞侵袭。6个视野中,Negative组(迁移细胞,167±7个),ORAI1干扰组(迁移细胞,71±20个),因此ORAI1干扰组的细胞侵袭实验中迁移细胞有减少(P<0.01)。
4.ⅢA期NSCLC患者肿瘤组织STIM1或ORAI1蛋白表达和预后的关系
4.1肿瘤组织STIM1或ORAI1表达和患者临床病理特征之间的关系
STIM1和ORAI1免疫组化阳性可见在肿瘤细胞胞浆中着色,部分胞膜着色。男性患者中有27例肿瘤组织STIM1低表达(64.3%,P=0.055)；鳞癌患者中有15例STIM1(?)表达(71.4%,P=0.084)；鳞癌患者中有18例ORAI1低表达(85.7%,P=0.067)。
4.2STIM1表达和ⅢA期NSCLC患者无疾病复发生存的相关性
男性NACLC患者中,STIM1低表达者预后有好于高表达者的趋势(21月vs12月,P=0.06)。鳞癌NSCLC患者中,STIM1低表达者预后有好于高表达者的趋势(33月vs9月,P=0.07)
研究结论
1.NSCLC肿瘤组织中可测到STIM1和ORAI1mRNA表达,并且腺癌中STIM1基因表达有增高的趋势,低分化肿瘤组织的ORAI1基因表达较高。
2.我们实验中发现A549细胞和16HBE细胞中存在着SOCE,并且SOCE部分参与这些细胞的增殖,而SOCE在A549细胞迁移和侵袭中起着重要的作用。
3.我们研究发现STIM1/oRAI1介导的SOCE没有参与人肺腺癌A549细胞的增殖,但在体外促进A549腺癌细胞的迁移和侵袭。
4.在鳞癌和男性ⅢA期NSCLC患者中,STIM1低表达可能预示着患者有较好的无疾病生存预后。
Background
Lung 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.
Objective
We 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 methods
1.24NSCLC 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.
Results
1. STIM1/ORAI1mRNA expressionin in NSCLC tumor samples
The 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 invasion
2.1SOCE in A549and16HBE
After 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 inhibitor
The 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 inhibitor
In 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 invasion
3.1Cell proliferation after silencing STIM1or0RAI1gene expression
We 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 expression
We 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 NSCLC
4.1. 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 level
Male 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)
Conclusions
1. 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.
肺癌是目前发病率和死亡率增长最快、对人类健康和生命威胁最大的恶性肿瘤。肺癌中80%是非小细胞肺癌(non-small cell lung cancer. NSCLC),病理上以鳞癌、腺癌和大细胞癌为主。因此研究肺癌的发生发展机制,寻找有效的治疗靶点,对于改善NSCLC预后,实施个体化治疗尤为重要。钙离子(Ca2+)作为第二信使,在细胞信号传导通路中起着重要的作用,调控细胞周期、分化、增殖和凋亡。近年来关于钙池操纵性钙通道(SOCC)的研究越来越多,由SOCC介导的钙池操纵性钙内流(SOCE)是非兴奋细胞例如上皮细胞产生Ca2+内流的主要方式。研究发现SOCE信号通路中的SITM1及ORAI1蛋白,由于其在介导SOCE中的作用而受到广泛关注。STIM1主要存在于细胞内质网膜上,在钙池消耗后,它作为一种“钙池信号感受蛋白”感受钙池充盈状态,并聚集成"puncta"向细胞膜移动靠近,而非嵌入到细胞膜中,此后STIM1通过羧基端与细胞膜上的SOCC通道蛋白如ORAI1的相互作用而开放钙通道,形成SOCE。STIM1和oRAI1介导的SOCE对上皮细胞的增殖、分化至关重要,因为多数肿瘤来源于上皮细胞,它们在肿瘤细胞中也陆续有相关的研究报道出现。NSCLC多来源于支气管上皮细胞,因此我们选择人肺腺癌细胞A549,研究SOCE在肺癌细胞中的表达情况；研究STIM1/ORAI1介导的SOCE对肺癌细胞的生物学行为如增殖、迁移和侵袭功能的影响；研究NSCLC患者手术肿瘤组织中STIM1或ORAI1表达和生存预后的关系。
研究目的
首先研究人NSCLC肿瘤组织中的SOCC通道STIM1和ORAI1的mRNA表达情况；其次在人肺腺癌细胞系A549中,研究SOCE与细胞增殖、迁移和侵袭的关系；然后研究STIM1和ORAI1蛋白在A549细胞增殖、迁移和侵袭功能中的作用；最后研究NSCLC患者手术肿瘤组织中STIM1或ORAI1蛋白表达和生存预后的关系。
研究方法
1、我们共收集2007-2009年NSCLC术后肿瘤组织和配对远癌正常肺组织(距肿瘤>5cm)24例,标本来自于广州医学院第一附属医院呼吸疾病研究所胸心外科。所有患者术前未行放化疗和其他抗肿瘤治疗。采用Trizol法提取组织总RNA,逆转录成cDNA,荧光定量PCR方法检测组织STIM1和ORAI1mRNA表达。
2、人肺腺癌A549细胞,人支气管上皮细胞16HBE购自中国科学院典型培养物保藏委员会细胞库。细胞增殖实验中,A549细胞和16HBE细胞中分别加入SOCE抑制剂SKF-96365或NiCl2。应用罗氏公司WST-1细胞增殖检测试剂盒检测24、48、72h的OD值,细胞增殖抑制率=1-实验组OD值／正常对照组OD值。A549细胞迁移和侵袭实验是应用Transwell小室法,迁移率=细胞迁移总数-上层未迁移细胞数／细胞总数,侵袭时检测迁移到Transwell小室的下室的细胞数。SOCE细胞钙功能测定是应用Fura-2Am荧光素方法检测,InCytIm2软件收集数据和分析。
3、我们设计合成siRNA, Nucleofector细胞核转染方法瞬时沉默STIMIIORAH基因表达,然后检测A549细胞的增殖、迁移和侵袭功能的改变,余检测方法同上。
4、我们应用S-P免疫组化方法检测70例ⅢA期NSCLC患者手术肿瘤组织STIM1和0RAI1蛋白表达,统计学分析和患者无疾病生存预后的关系。
研究结果
1.NSCLC肿瘤组织中STIM1/ORAI1mRNA的表达
24例患者肿瘤组织STIM1mRNA相对表达量中位值0.84(0.19-3.4),1.09±0.82(X±S)；正常组织STIM1mRNA相对表达量中位值0.83(0.33-2.6)0.91±0.51(P>0.05)。17例患者肿瘤组织ORAI1mRNA相对表达量中位值0.95(0.31-4.9),1.51±1.46；正常肺组织ORAI1mKNA相对表达量中位值0.76(0.31-4.0),1.0±0.92(P>0.05)。在亚组分析中,我们发现腺癌和鳞癌之间的STIM1、ORAI1mKNA的表达无统计学差异(P>0.05),但腺癌的STIM1表达有高于鳞癌的趋势(P=0.055)；低分化肿瘤组织中ORAI1表达要高于分化较好的肿瘤和正常肺组织(P=0.028<0.05),而STIM1却没有类似发现。
2.SOCE对A549肺腺癌细胞的增殖、迁移和侵袭的影响
2.1A549和16HBE细胞的SOCE
在含钙PSS溶液灌流后,不同浓度的SKF-96365和NiCl2都可以减少钙内流,并呈浓度依赖性。我们选取8-14个细胞,相对于未处理过的A549细胞(钙内流峰值Δ[Ca2+]i,482±36nM), SKF-96365处理过的细胞出现△[Ca2+]i下降(P<0.001)：其中SKF-963651μM (372±17nM);10μM (268±26nM);50μM (188±23nM)。相对于未处理的A549细胞(钙内流峰值Δ[Ca2+]i,482±36nM), NiCl2处理过的细胞出现△[Ca2+]i下降(P<0.001)：其中NiCl250μM(268±33nM);200μM (250±55nM);500μM(208±47nM)。
在含钙PSS溶液灌流后,低浓度的SKF-96365增加16HBE细胞钙内流,但高浓度的SKF-96365才减少细胞钙内流。我们选取9-11个细胞,相对于未处理过的16HBE细胞(钙内流峰值Δ[Ca2+]i,290±53nM),低浓度SKF-96365处理过的细胞出现△[Ca2+]i上升(P<0.001)：其中SKF-963651μM (743±93nM);10μM (510±32nM),但高浓度SKF-9636550μM处理后细胞钙内流减少(180±29nM)。相对于未处理过的16HBE细胞(钙内流峰值Δ[Ca2+]i,460±45nM), NiCl2处理过的细胞出现△[Ca2+]i下降(P<0.001)：其中NiCl2200μM (337±37nM);500μM(173±28nM);1000μM(58±6.1nM)。
2.2SOCE抑制剂处理后的A549和16HBE细胞增殖实验
我们首先选择不同浓度SKF-96365处理A549细胞,48h后观察细胞增殖抑制率,结果显示：SKF-963651μM(33%±5%);10μM(46%±4%);50μM(58%±1%),因此我们选择SKF-9636510μM进行下一步的实验。A549细胞经过SKF-9636510μM处理24h、48h和72h,我们发现不同时间点细胞的增殖受到明显的抑制,在24h、48h和72h的抑制率分别为20%±3%,52%±4%,73%±6%。16HBE细胞经过SKF-9636510μM处理24h、48h和72h,发现不同时间点细胞的增殖受到抑制,在24h、48h和72h的抑制率分别为14%±4%,38%±7%；67%±3%。
我们首先选择不同浓度NiCl2处理A549细胞,48h后观察细胞增殖抑制率,结果显示：NiCl2200μM (44%±3%);500μM (50%±4%);1000μM(64%±10%),因此我们选择NiCl2500μM进行下一步的实验。A549细胞经过NiCl2500μM处理24h、48h和72h,我们发现不同时间点细胞的增殖受到抑制,在24h、48h和72h的抑制率分别为12%±4%,50%±4%,62%±9%。16HBE细胞经过NiCl2500μM处理24h、48h和72h,我们发现不同时间点细胞的增殖受到抑制,在24h、48h和72h的抑制率分别为14%±4%,43%±3%,63%±4%。
2.3SOCE抑制剂处理后的A549细胞的迁移和侵袭
我们在预实验中发现A549细胞有迁移能力,而16HBE细胞无迁移能力。我们发现不同浓度的SKF-96365和NiCl2可以明显抑制A549细胞的迁移。相对于未处理过的A549细胞(迁移率,49%±3%),SKF-96365处理后的A549细胞的迁移率降低：SKF-963651μM(20%±3%);10μM(10%±2%);50μM(8%±2%)(P<0.001)。相对于未处理过的A549细胞(迁移率,47%±2%),NiCl250μM (32%±6%);200μM (30%±5%);500μM (19%±4%);1000μM(11%±4%)(P<0.001)。实验中,我们还发现低浓度的SKF-963651μM和NiCl250μM都可以明显抑制A549细胞的迁移。
不同浓度的SKF-96365和NiCl2可以明显抑制A549细胞的侵袭。相对于未处理过的A549细胞(6个视野迁移细胞,503±45个),SKF-96365处理后的A549细胞的迁移细胞减少：SKF-963651μM(210±36个)；10μM(110±26个)；50μM(20±5个)(P<0.001)。相对于未处理过的A549细胞(6个视野迁移细胞,503±45个),NiCl2200μM(396±55个)；500μM(320±53个)；1000μM(100±30个),其中NiCl2500μM和1000μM处理后的迁移细胞相对于未处理组减少(P<0.01)
3. STIM1/ORAI1在A549细胞增殖、迁移和侵袭功能中的作用
3.1siRNA瞬时沉默A549细胞STIM1/ORAI1基因表达
我们应用siRNA沉默A549细胞STIM1基因表达,48h基因瞬时沉默效率最高,可达84%,72h蛋白瞬时沉默效率最高,约70%。我们应用siRNA沉默A549细胞ORAI1基因表达,24h基因瞬时沉默效率最高,可达91%,48h蛋白瞬时沉默效率最高,约80%。
3.2A549细胞STIM1/ORAI1基因表达沉默后的SOCE
我们在瞬时干扰后72h进行钙功能的测定。相对于Negative干扰组(钙内流峰值Δ[Ca2+]i,284±52nM), STIM1干扰组出现了SOCE的下降,约1/3(钙内流峰值Δ[Ca2+]i,194±28nM)(P<0.001)。我们在瞬时干扰后48小时进行钙功能的测定。相对于Negative干扰组(钙内流峰值Δ[Ca2+]i,145±47nM) ORAI1干扰组出现了SOCE的下降,约1/3(钙内流峰值Δ[Ca2+]i,92±24nM)(P<0.05)。
3.3沉默A549细胞STIM1/ORAI1基因表达后的细胞增殖试验
我们发现siRNA瞬时沉默STIM1表达后,A549细胞增殖功能没有明显改变。STIM1或ORAI1siRNA干扰后A549细胞24-96h生长曲线和Negative干扰组无差异。
3.4沉默A549细胞STIM1/ORAI1基因表达后的细胞迁移和侵袭试验
我们发现siRNA瞬时沉默A549细胞STIM1表达后,72h后我们应用Transwell小室法检测细胞迁移率。相对于Negative组(迁移率,38%±2%)STIM1干扰组(迁移率,14%±4%),因此STIM1干扰组的细胞迁移率有下降(P<0.01)。siRNA瞬时沉默A549细胞STIM1表达后,72h后我们应用Transwell小室法检测细胞侵袭。6个视野中,Negative组(迁移细胞,246±23个),STIM1干扰组(迁移细胞,80±11个),因此STIM1干扰组的细胞侵袭实验中迁移细胞有减少(P<0.001)。siRNA瞬时沉默A549细胞ORAll表达后,48h后我们应用Transwell小室法检测细胞迁移率。相对于Negative组(迁移率,30%±12%),ORAI1干扰组(迁移率,12%±5%),与对照组相比ORAIl干扰组的细胞迁移率有下降的趋势,虽然无统计学意义(P=0.084)。siRNA瞬时沉默A549细胞ORAI1表达后,48小时后我们应用Transwell小室法检测细胞侵袭。6个视野中,Negative组(迁移细胞,167±7个),ORAI1干扰组(迁移细胞,71±20个),因此ORAI1干扰组的细胞侵袭实验中迁移细胞有减少(P<0.01)。
4.ⅢA期NSCLC患者肿瘤组织STIM1或ORAI1蛋白表达和预后的关系
4.1肿瘤组织STIM1或ORAI1表达和患者临床病理特征之间的关系
STIM1和ORAI1免疫组化阳性可见在肿瘤细胞胞浆中着色,部分胞膜着色。男性患者中有27例肿瘤组织STIM1低表达(64.3%,P=0.055)；鳞癌患者中有15例STIM1(?)表达(71.4%,P=0.084)；鳞癌患者中有18例ORAI1低表达(85.7%,P=0.067)。
4.2STIM1表达和ⅢA期NSCLC患者无疾病复发生存的相关性
男性NACLC患者中,STIM1低表达者预后有好于高表达者的趋势(21月vs12月,P=0.06)。鳞癌NSCLC患者中,STIM1低表达者预后有好于高表达者的趋势(33月vs9月,P=0.07)
研究结论
1.NSCLC肿瘤组织中可测到STIM1和ORAI1mRNA表达,并且腺癌中STIM1基因表达有增高的趋势,低分化肿瘤组织的ORAI1基因表达较高。
2.我们实验中发现A549细胞和16HBE细胞中存在着SOCE,并且SOCE部分参与这些细胞的增殖,而SOCE在A549细胞迁移和侵袭中起着重要的作用。
3.我们研究发现STIM1/oRAI1介导的SOCE没有参与人肺腺癌A549细胞的增殖,但在体外促进A549腺癌细胞的迁移和侵袭。
4.在鳞癌和男性ⅢA期NSCLC患者中,STIM1低表达可能预示着患者有较好的无疾病生存预后。
Background
Lung 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.
Objective
We 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 methods
1.24NSCLC 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.
Results
1. STIM1/ORAI1mRNA expressionin in NSCLC tumor samples
The 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 invasion
2.1SOCE in A549and16HBE
After 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 inhibitor
The 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 inhibitor
In 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 invasion
3.1Cell proliferation after silencing STIM1or0RAI1gene expression
We 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 expression
We 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 NSCLC
4.1. 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 level
Male 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)
Conclusions
1. 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.
引文
1. Roos J, DiGregorio PJ, Yeromin AV, et al. STIM1, an essential and conserved component of store-operated Ca2+channel function. J Cell Biol 2005;169(3):435-45.
2. Liou J, Kim ML, Heo WD, et al. STIM is a Ca2+sensor essential for Ca2+-store depletion-triggered Ca2+influx. Curr Biol 2005;15(13):1235-41.
3. Zhang SL, Yu Y, Roos J, et al. STIM1 is a Ca2+sensor that activates CRAC channels and migrates from the Ca2+ store to the plasma membrane. Nature 2005;437(7060):902-5.
4. Vig M, Kinet JP. Calcium signaling in immune cells. Nat Immunol 2009;10(1):21-7.
5. Hewavitharana T, Deng X, Soboloff J, et al. Role of STIM and ORAI proteins in the store-operated calcium signaling pathway. Cell Calcium 2007;42(2):173-82.
6. Lu W, Wang J, Shimoda LA, et al. Differences in STIM1 and TRPC expression in proximal and distal pulmonary arterial smooth muscle are associated with differences in Ca2+responses to hypoxia. Am J Physiol Lung Cell Mol Physiol 2008;295(1):L104-13.
7. Abdullaev IF, Bisaillon JM, Potier M, et al. STIM1 and ORAI1 mediate CRAC currents and store-operated calcium entry important for endothelial cell proliferation. Circ Res 2008:103(11):1289-1299.
8. Agrotis A, Koulis C. STIM1:a new therapeutic target in occlusive vascular disease? Cardiovasc Res 2009:81 (4):627-8.
9. Vig M, Peinelt C, Beck A, et al. CRACM1 is a plasma membrane protein essential for store-operated Ca2+entry. Science 2006;312(5777):1220-3.
10. Liao Y, Plummer NW, George MD, et al. A role for Orai in TRPC-imediated Ca2+entry suggests that a TRPC:Orai complex may mediate store and receptor operated Ca2+entry. Proc Natl Acad Sci U S A 2009;106(9):3202-6.
11. Soboloff J, Spassova MA, Tang XD, et al. ORAI1 and STIM reconstitute store-operated calcium channel function. J Biol Chem 2006;281(30):20661-5.
12. Mercer JC, Dehaven WI, Smyth JT, et al. Large store-operated calcium selective currents due to co-expression of ORAI1 or Orai2 with the intracellular calcium sensor, STIM1. J Biol Chem 2006;281(34):24979-90.
13. Liao Y, Erxleben C, Yildirim E, et al. Orai proteins interact with TRPC channels and confer responsiveness to store depletion. Proc Natl Acad Sci U S A. 2007;104(11):4682-7.
14. Thebault S, Flourakis M, Vanoverberghe K, et al. Differential Role of Transient Receptor Potential Channels in Ca2+Entry and Proliferation of Prostate Cancer Epithelial Cells. Cancer Res 2006;66(4):2038-47.
15. Mercer JC, DeHaven WI, Smyth JT, et al. Large store-operated calcium-selected currents due to co-expression of ORAI1 or orai2 with the intracellular calcium sensor, STIM1. J Biol Chem 2006;281:249-79.
16. Thebault S, Flourakis M, Vanoverberghe K, et.al. Differential Role of Transient Receptor Potential Channels in Ca2+Entry and Proliferation of Prostate Cancer Epithelial Cells.Cancer Res 2006; 66(4):2038-47.
17. El Boustany C, Bidaux G, Enfissi A.et al. Capacitative calcium entry and transient receptor potential canonical 6 expression control human hepatoma cell proliferation. Hepatology 2008;47(6):2068-77.
18. Yang S, Zhang JJ, Huang XY. ORAI1 and STIM1 Are Critical for Breast Tumor Cell Migration and Metastasis. Cancer Cell 2009; 15(2):124-34.
19. Suyama E, Wadhwa R, Kaur K, et al. Identification of metastasis-related genes in a mouse model using a library of randomized ribozymes. J Biol Chem 2004;279(37):38083-6.
20.张奇,何建行,卢文菊等.经典瞬时受体电位通道蛋白在人非小细胞肺癌组织中的表达.中国肺癌杂志；2010；13(6)：612-6.
21. Gudermann T, Roelle S. Calcium-dependent growth regulation of small cell lung cancer cells by neuropeptides. Endocr Relat Cancer 2006; 13(4):1069-84.
22. Obukhov AG, Nowycky MC. TRPC4 can be activated by G-protein-coupled receptors and provides sufficient Ca2+to trigger exocytosis in neuroendocrine cells. J Biol Chem 2002:277(18):16172-8.
23. Gailly P, Colson-Van Schoor M. Involvement of trp-2 protein in store-operated influx of calcium in fibroblasts. Cell Calcium 2001;30(3):157-65.
24. Jungnickel MK, Marrero H, Birnbaumer L, et al. Trp2 regulated entry of Ca2+ into mouse wperm triggered by egg ZP3. Nat Cell Biol 2001;3(5):499-502.
25. Sanchez-Hernandez Y, Laforenza U, Bonetti E, et al. Store-operated Ca(2+) entry is expressed in human endothelial progenitor cells. Stem Cells Dev. 2010;19(12):1967-81.
26. Shi Y, Song M, Guo R, et al. Knockdown of stromal interaction molecule 1 attenuates hepatocyte growth factor-induced endothelial progenitor cell proliferation. Exp Biol Med (Maywood).2010;235(3):317-25.
27. Wang C, Li JF, Zhao L, et al. Inhibition of SOC/Ca2+/NFAT pathway is involved in the anti-proliferative effect of sildenafil on pulmonary artery smooth muscle cells. Respir Res.2009;10(l):123.
28. Ahmmed GU, Mehta D, Vogel S, et al. Protein kinase Calpha phosphorylates the TRPC1 channel and regulates store-operated Ca2 entry in endothelial cells. J Biol Chem.2004;279:20941-49.
29. Cioffi DL, Wu S, Alexeyev M, et al. Activation of the endothelial store-operated ISOC Ca2 channel requires interaction of protein 4.1 with TRPC4. Circ Res 2005:97:1164-72.
30. Dong H, Shim KN, Li JM, et al. Molecular mechanisms underlying Ca2+-mediated motility of human pancreatic duct cells. Am J Physiol Cell Physiol 2010;299(6):C1493-503.
31. Wang Y, Yue D, Li K, et al. The role of TRPC6 in HGF-induced cell proliferation of human prostate cancer DU145 and PC3 cells. Asian J Androl 2010;12(6):841-52.
32. Motiani RK, Abdullaev IF, Trebak M. A novel native store-operated calcium channel encoded by Orai3:selective requirement of Orai3 versus ORAI1 in estrogen receptor-positive versus estrogen receptor-negative breast cancer cellsJ Biol Chem.2010;285(25):19173-83.
33. Flourakis M, Lehen'kyi V, Beck B, et al. ORAI1 contributes to the establishment of an apoptosis-resistant phenotype in prostate cancer cells. Cell Death Dis 2010;1(9):e75.
34. Franco SJ, Rodgers MA, Perrin BJ, et al. Calpain-mediated proteolysis of talin regulates adhesion dynamics[J]. Nature Cell Biology 2004,6(10):977-83.
35. Chen YF, Chiu WT, Chen YT, et al. Calcium store sensor stromal-interaction molecule 1-dependent signaling plays an important role in cervical cancer growth, migration, and angiogenesis. Proc Natl Acad Sci U S A 2011;108(37):15225-30.
1 Lipskaia L, Hulot, Lopre AM. Role of sarco/endoplasmic reticulum calcium content and calcium ATPase activity in the control of cell growth and proliferation. Pflugers Arch 2009;457:673-685.
2 Mattson MP, Chan SL. Calcium orchestrates apoptosis. Nat Cell Biol 2003;5:1041-1043.
3 Orrenius S, Zhivotovsky B, Nicotera P. Regulation of cell death:the calcium-apoptosis link. Nat Rev Mol Cell Bil 2003;4:552-565.
4 Berridge MJ, Lipp P, Bootman MD. The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 2000;1:11-21.
5 Carafoli E. Calcium signaling:a tale for all seasons. Proc Natl Acad Sci USA 2002;99:1115-1122.
6 Blaustein MP and Lederer WJ. Sodium/calcium exchage:its physiological implications. Physiol Rev 1999:79:763-854.
7 Lytton J, Li XF, Dong H, Kraev A. K+-dependent Na+/Ca2+exchangers in the brain. Ann N Y Acad Sci 2002;976:382-393.
8 Hryshko LV. Tissue-specific modes of Na/Ca exchanger regulation. Ann N Y Acad Sci 2002;976:166-175.
9 Berridge MJ, Bootman MD, Roderick HL. Calcium signalling:dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 2003:4:517-529.
10 Clapham DE. Calcium signalling. Cell 2007; 131:1047-1058.
11 Parekh AB, Putney JW. Store-operated calcium channels. Physiol Rev 2005;85:757-810.
12 Abdullaev IF, Bisaillon JM, Potier M, Gonzalez JC, Motiani RK, et al. Stiml and Orail mediate CRAC currents and store-operated calcium entry important for endothelial cell proliferation. Circ Res 2008;103:1289-1299.
13 Zhang SL, Yu Y, Roos J, Kozak JA, Deerinck TJ, Ellisman MH, et al. Stiml is Ca2+sensor that activates CRAC channels and migrates from the Ca2+store to the plasma membrane. Nature 2005;437:902-905.
14 Peel SE, Liu B, Hall IP. ORAI and store-operated calcium influx in human airway smooth muscle cells. Am J Respir Cell Mol Biol 2008;38:744-749.
15 Liao Y, Erxleben C, Yildirim E. Orai proteins interact with TRPC channels and confer responsiveness to store depletion. Proc Natl Acad Sci U S A 2007;104:4682-4687.
16 Liao Y, Erxleben C, Abramowitz J, Flockerzi V, Zhu MX, et al. Functional interactions among Orail, TRPCs, and STIM1 suggest a S TIM-regulated heteromeric Orai/TRPC modes for SOCE/Icrac channels. Natl Acad Sci U S A 2008;105:2895-2900.
17 Jaffe LF. A calcium-based theory of carcinogenesis. Adv Cancer Res 2005:94:231-263.
18 Guilbert A, Dhennin-Duthille I, Hiani YE, et al. Expression of TRPC6 channels in human epithelial breast cancer cells. BMC Cancer 2008;8:125.
19 El Hiani Y, Lehen'kyi V, Ouadid-Ahidouch HA, et al. Activation of the calcium-sensing receptor by high calcium induced breast cancer cell proliferation and TRPC1 cation channel over-expression potentially through EGFR pathways. Arch Biochem Biophys 2009;486:58-63.
20 Pigozzi D, Ducret T, Tajeddine N, et al. Calcium store contents control the expression of TRPC1, TRPC3 and TRPV6 proteins in LNCaP prostate cancer cell line. Cell Calcium 2006;39:401-405.
21 El Boustany C, Bidaux G, Enfissi A, et al. Capacitative calcium entry and transient receptor potential canonical 6 expression control human hepatoma cell proliferation. Hepatoloty 2008;47:2068-2077.
22 Thebault S, Flourakis M, Vanoverberghe K, et al. Differential role of transient receptor potential channels in Ca2+entry and proliferation of prostate cancer epithelial cells. Cancer Res 2006;66:2038-2047.
23 Yang S, Zhang JJ, Huang XY. Orai1 and STIM1 are critical for breast tumor cell migration and metastsis. Cancer Cell 2009; 15:124-134.
24 El Boustany C, Bidaux G, Enfissi A, et al. Capacitative calcium entry and transient receptor potential canonical 6 expression control human hepatoma cell proliferation. Hepatology 2008;47:2068-2077.
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2. Liou J, Kim ML, Heo WD, et al. STIM is a Ca2+sensor essential for Ca2+-store depletion-triggered Ca2+influx. Curr Biol 2005;15(13):1235-41.
3. Zhang SL, Yu Y, Roos J, et al. STIM1 is a Ca2+sensor that activates CRAC channels and migrates from the Ca2+ store to the plasma membrane. Nature 2005;437(7060):902-5.
4. Vig M, Kinet JP. Calcium signaling in immune cells. Nat Immunol 2009;10(1):21-7.
5. Hewavitharana T, Deng X, Soboloff J, et al. Role of STIM and ORAI proteins in the store-operated calcium signaling pathway. Cell Calcium 2007;42(2):173-82.
6. Lu W, Wang J, Shimoda LA, et al. Differences in STIM1 and TRPC expression in proximal and distal pulmonary arterial smooth muscle are associated with differences in Ca2+responses to hypoxia. Am J Physiol Lung Cell Mol Physiol 2008;295(1):L104-13.
7. Abdullaev IF, Bisaillon JM, Potier M, et al. STIM1 and ORAI1 mediate CRAC currents and store-operated calcium entry important for endothelial cell proliferation. Circ Res 2008:103(11):1289-1299.
8. Agrotis A, Koulis C. STIM1:a new therapeutic target in occlusive vascular disease? Cardiovasc Res 2009:81 (4):627-8.
9. Vig M, Peinelt C, Beck A, et al. CRACM1 is a plasma membrane protein essential for store-operated Ca2+entry. Science 2006;312(5777):1220-3.
10. Liao Y, Plummer NW, George MD, et al. A role for Orai in TRPC-imediated Ca2+entry suggests that a TRPC:Orai complex may mediate store and receptor operated Ca2+entry. Proc Natl Acad Sci U S A 2009;106(9):3202-6.
11. Soboloff J, Spassova MA, Tang XD, et al. ORAI1 and STIM reconstitute store-operated calcium channel function. J Biol Chem 2006;281(30):20661-5.
12. Mercer JC, Dehaven WI, Smyth JT, et al. Large store-operated calcium selective currents due to co-expression of ORAI1 or Orai2 with the intracellular calcium sensor, STIM1. J Biol Chem 2006;281(34):24979-90.
13. Liao Y, Erxleben C, Yildirim E, et al. Orai proteins interact with TRPC channels and confer responsiveness to store depletion. Proc Natl Acad Sci U S A. 2007;104(11):4682-7.
14. Thebault S, Flourakis M, Vanoverberghe K, et al. Differential Role of Transient Receptor Potential Channels in Ca2+Entry and Proliferation of Prostate Cancer Epithelial Cells. Cancer Res 2006;66(4):2038-47.
15. Mercer JC, DeHaven WI, Smyth JT, et al. Large store-operated calcium-selected currents due to co-expression of ORAI1 or orai2 with the intracellular calcium sensor, STIM1. J Biol Chem 2006;281:249-79.
16. Thebault S, Flourakis M, Vanoverberghe K, et.al. Differential Role of Transient Receptor Potential Channels in Ca2+Entry and Proliferation of Prostate Cancer Epithelial Cells.Cancer Res 2006; 66(4):2038-47.
17. El Boustany C, Bidaux G, Enfissi A.et al. Capacitative calcium entry and transient receptor potential canonical 6 expression control human hepatoma cell proliferation. Hepatology 2008;47(6):2068-77.
18. Yang S, Zhang JJ, Huang XY. ORAI1 and STIM1 Are Critical for Breast Tumor Cell Migration and Metastasis. Cancer Cell 2009; 15(2):124-34.
19. Suyama E, Wadhwa R, Kaur K, et al. Identification of metastasis-related genes in a mouse model using a library of randomized ribozymes. J Biol Chem 2004;279(37):38083-6.
20.张奇,何建行,卢文菊等.经典瞬时受体电位通道蛋白在人非小细胞肺癌组织中的表达.中国肺癌杂志；2010；13(6)：612-6.
21. Gudermann T, Roelle S. Calcium-dependent growth regulation of small cell lung cancer cells by neuropeptides. Endocr Relat Cancer 2006; 13(4):1069-84.
22. Obukhov AG, Nowycky MC. TRPC4 can be activated by G-protein-coupled receptors and provides sufficient Ca2+to trigger exocytosis in neuroendocrine cells. J Biol Chem 2002:277(18):16172-8.
23. Gailly P, Colson-Van Schoor M. Involvement of trp-2 protein in store-operated influx of calcium in fibroblasts. Cell Calcium 2001;30(3):157-65.
24. Jungnickel MK, Marrero H, Birnbaumer L, et al. Trp2 regulated entry of Ca2+ into mouse wperm triggered by egg ZP3. Nat Cell Biol 2001;3(5):499-502.
25. Sanchez-Hernandez Y, Laforenza U, Bonetti E, et al. Store-operated Ca(2+) entry is expressed in human endothelial progenitor cells. Stem Cells Dev. 2010;19(12):1967-81.
26. Shi Y, Song M, Guo R, et al. Knockdown of stromal interaction molecule 1 attenuates hepatocyte growth factor-induced endothelial progenitor cell proliferation. Exp Biol Med (Maywood).2010;235(3):317-25.
27. Wang C, Li JF, Zhao L, et al. Inhibition of SOC/Ca2+/NFAT pathway is involved in the anti-proliferative effect of sildenafil on pulmonary artery smooth muscle cells. Respir Res.2009;10(l):123.
28. Ahmmed GU, Mehta D, Vogel S, et al. Protein kinase Calpha phosphorylates the TRPC1 channel and regulates store-operated Ca2 entry in endothelial cells. J Biol Chem.2004;279:20941-49.
29. Cioffi DL, Wu S, Alexeyev M, et al. Activation of the endothelial store-operated ISOC Ca2 channel requires interaction of protein 4.1 with TRPC4. Circ Res 2005:97:1164-72.
30. Dong H, Shim KN, Li JM, et al. Molecular mechanisms underlying Ca2+-mediated motility of human pancreatic duct cells. Am J Physiol Cell Physiol 2010;299(6):C1493-503.
31. Wang Y, Yue D, Li K, et al. The role of TRPC6 in HGF-induced cell proliferation of human prostate cancer DU145 and PC3 cells. Asian J Androl 2010;12(6):841-52.
32. Motiani RK, Abdullaev IF, Trebak M. A novel native store-operated calcium channel encoded by Orai3:selective requirement of Orai3 versus ORAI1 in estrogen receptor-positive versus estrogen receptor-negative breast cancer cellsJ Biol Chem.2010;285(25):19173-83.
33. Flourakis M, Lehen'kyi V, Beck B, et al. ORAI1 contributes to the establishment of an apoptosis-resistant phenotype in prostate cancer cells. Cell Death Dis 2010;1(9):e75.
34. Franco SJ, Rodgers MA, Perrin BJ, et al. Calpain-mediated proteolysis of talin regulates adhesion dynamics[J]. Nature Cell Biology 2004,6(10):977-83.
35. Chen YF, Chiu WT, Chen YT, et al. Calcium store sensor stromal-interaction molecule 1-dependent signaling plays an important role in cervical cancer growth, migration, and angiogenesis. Proc Natl Acad Sci U S A 2011;108(37):15225-30.
1 Lipskaia L, Hulot, Lopre AM. Role of sarco/endoplasmic reticulum calcium content and calcium ATPase activity in the control of cell growth and proliferation. Pflugers Arch 2009;457:673-685.
2 Mattson MP, Chan SL. Calcium orchestrates apoptosis. Nat Cell Biol 2003;5:1041-1043.
3 Orrenius S, Zhivotovsky B, Nicotera P. Regulation of cell death:the calcium-apoptosis link. Nat Rev Mol Cell Bil 2003;4:552-565.
4 Berridge MJ, Lipp P, Bootman MD. The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 2000;1:11-21.
5 Carafoli E. Calcium signaling:a tale for all seasons. Proc Natl Acad Sci USA 2002;99:1115-1122.
6 Blaustein MP and Lederer WJ. Sodium/calcium exchage:its physiological implications. Physiol Rev 1999:79:763-854.
7 Lytton J, Li XF, Dong H, Kraev A. K+-dependent Na+/Ca2+exchangers in the brain. Ann N Y Acad Sci 2002;976:382-393.
8 Hryshko LV. Tissue-specific modes of Na/Ca exchanger regulation. Ann N Y Acad Sci 2002;976:166-175.
9 Berridge MJ, Bootman MD, Roderick HL. Calcium signalling:dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 2003:4:517-529.
10 Clapham DE. Calcium signalling. Cell 2007; 131:1047-1058.
11 Parekh AB, Putney JW. Store-operated calcium channels. Physiol Rev 2005;85:757-810.
12 Abdullaev IF, Bisaillon JM, Potier M, Gonzalez JC, Motiani RK, et al. Stiml and Orail mediate CRAC currents and store-operated calcium entry important for endothelial cell proliferation. Circ Res 2008;103:1289-1299.
13 Zhang SL, Yu Y, Roos J, Kozak JA, Deerinck TJ, Ellisman MH, et al. Stiml is Ca2+sensor that activates CRAC channels and migrates from the Ca2+store to the plasma membrane. Nature 2005;437:902-905.
14 Peel SE, Liu B, Hall IP. ORAI and store-operated calcium influx in human airway smooth muscle cells. Am J Respir Cell Mol Biol 2008;38:744-749.
15 Liao Y, Erxleben C, Yildirim E. Orai proteins interact with TRPC channels and confer responsiveness to store depletion. Proc Natl Acad Sci U S A 2007;104:4682-4687.
16 Liao Y, Erxleben C, Abramowitz J, Flockerzi V, Zhu MX, et al. Functional interactions among Orail, TRPCs, and STIM1 suggest a S TIM-regulated heteromeric Orai/TRPC modes for SOCE/Icrac channels. Natl Acad Sci U S A 2008;105:2895-2900.
17 Jaffe LF. A calcium-based theory of carcinogenesis. Adv Cancer Res 2005:94:231-263.
18 Guilbert A, Dhennin-Duthille I, Hiani YE, et al. Expression of TRPC6 channels in human epithelial breast cancer cells. BMC Cancer 2008;8:125.
19 El Hiani Y, Lehen'kyi V, Ouadid-Ahidouch HA, et al. Activation of the calcium-sensing receptor by high calcium induced breast cancer cell proliferation and TRPC1 cation channel over-expression potentially through EGFR pathways. Arch Biochem Biophys 2009;486:58-63.
20 Pigozzi D, Ducret T, Tajeddine N, et al. Calcium store contents control the expression of TRPC1, TRPC3 and TRPV6 proteins in LNCaP prostate cancer cell line. Cell Calcium 2006;39:401-405.
21 El Boustany C, Bidaux G, Enfissi A, et al. Capacitative calcium entry and transient receptor potential canonical 6 expression control human hepatoma cell proliferation. Hepatoloty 2008;47:2068-2077.
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