瞬时受体电位通道TRPM7在人鼻咽癌细胞中的表达及其对细胞迁移能力影响的初步研究
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摘要
研究背景和目的：
目前恶性肿瘤的治疗之所以失败的原因,一是耐药性,二是无法防治肿瘤的转移。恶性肿瘤在诊断时局部淋巴结转移发生率为1／3－1／4。而肿瘤致死亡的主要原因就是肿瘤细胞的转移,90%肿瘤患者最终死于转移。但当前人们对肿瘤转移机制缺乏系统而深入的认识；现行临床治疗手段对抑制肿瘤转移基本无效；故寻找肿瘤转移相关因子,揭示肿瘤转移的分子机制便成为当今肿瘤研究面临的最大挑战及国际研究的热点。
肿瘤细胞的侵袭转移涉及肿瘤细胞及间质细胞和基质之间的多个步骤和分子反应。目前肿瘤转移步骤的模式广为人们接受的是Liotta提出的癌细胞侵袭转移的三步骤假说：1.肿瘤细胞之间的解黏附和肿瘤细胞与ECM之间的黏附；2.侵足的形成与ECM的降解：肿瘤细胞和宿主细胞分泌的蛋白水解酶,使肿瘤细胞周围的发生降解；3.运动,肿瘤细胞被生长因子及趋化因子诱导,向纵深运动。值得一提的是,虽然上述个阶段涉及到相对特异的分子机制,但是侵袭转移过程中各阶段之间的分子存在交叉和相互作用,因此侵袭转移的阶段和分子功能划分只是相对的。
已有研究证明,正常组织细胞在特殊情况下也具备迁移能力,如胚胎发育、创伤愈合、免疫细胞。而肿瘤细胞由正常细胞经过演变,形成强于正常组织细胞的转移能力。肿瘤细胞虽然具备自身特点,但由于来源于正常组织细胞,因此在很多方面还是原有功能的沿用或是增强。而当今一些肿瘤转移重大发现采用了类比的方式,跨学科从生理学中寻找对于正常细胞迁移的重要分子,试图找到对于肿瘤细胞至关重要的转移相关因子。比如肿瘤生物学最新研究表明：在胚胎发育中起重要作用的上皮至间质转换(Epithelial-mesenchymal transition, EMT)便是肿瘤转移的重要机制之一。我们的研究思路正是采用这种交叉学科间的类比思维,试图通过寻找在正常细胞中证实已与细胞迁移密切相关的因子,同时收集5大瘤种10株转移能力不同的肿瘤细胞,从基因和蛋白水平比较,而非采取常规的高通量基因蛋白芯片,试图找到与肿瘤细胞转移能力相关的分子而进一步对其进行深入研究。经过文献调研,我们认为瞬时受体电位通道家族之一的TRPM7可能与肿瘤细胞的迁移相关,初步证据如下：
瞬时受体电位通道(TRP channels)是位于细胞膜上的一类重要的阳离子通道超家族。TRP通道分为：TRPC、TRPV、TRPM、TRPA、TRPP和TRPML6大亚家族。TRPM (Melastatin)亚家族包括TRPM1-8,共8种亚型。TRP通道为非选择性阳离子通道,TRPM7通道主要通过的离子为钙离子、钠离子,其还可通过镁、锌、锰等很多二价离子。
北京大学程和平等运用共聚焦显微成像技术,发现迁移成纤维细胞头部存在大量微小而短暂的钙信号事件,即“钙闪烁”现象,并证明钙闪烁起着掌控细胞运动“方向舵”的作用。实验条件下,钙闪烁被抑制的细胞可以维持直线运动,但完全丧失了定向和转弯的能力。进而提出了“钙闪烁引导细胞定向迁移”的新观点：在外界趋化因子梯度诱导下,钙闪烁发放呈现不对称特性,即趋化因子浓度高的一侧,钙闪烁更为活跃,驱动细胞转向此侧,从而精确地调控细胞的定向迁移。而在文章中作者通过实验验证：在所有可能的TRP通道中,TRPM7通道是唯一调控迁移细胞前端的“钙闪烁”现象的通道蛋白,即TRPM7才是真正掌控细胞运动的“方向舵”,并以此来指引细胞跟随趋化因子梯度进而有目的的定向迁移。
此外,Kristopher Clark等发现在小鼠N1E-115神经母细胞瘤细胞中,TRPM7介导缓激肽(BK)诱导的钙内流；同时过表达TRPM7可以促进该细胞的伸展并增强细胞与基质之间的粘附；此外TRPM7的激活还可以导致激酶依赖的足体的形成。而对于足体,目前研究表明：正常细胞的足体与肿瘤细胞的侵足关系密切,人们推测足体是侵入性伪足的前体,在一定条件下形成稳定的侵入性伪足。足体在生理物质上显示出降解细胞外基质的功能。我们也可以由此推测：TRPM7也可能在肿瘤细胞中通过导致侵足的形成继而增强肿瘤细胞的侵袭能力。另外,还有研究表明：TRPM7可与细胞膜表面的马达蛋白myosinⅡ相互作用,从而通过调控细胞骨架解除细胞与基质的粘附进而影响细胞的伸展。
综上,TRPM7已被证明的一系列细胞迁移相关的作用与肿瘤细胞迁移、侵袭的各个阶段均具有相关性,故我们大胆推测：TRPM7可能是肿瘤细胞侵袭转移的重要调控分子。
方法：
1.肿瘤细胞株的选取及TRPM7基因／蛋白水平表达的差异检测
选取分属鼻咽癌、乳腺癌、肺癌、大肠癌、肝癌等5大瘤种中已公认转移能力不同的10株肿瘤细胞株为研究对比对象。运用RT-PCR和Western Blot从基因和蛋白两个表达水平检测不同瘤种的肿瘤细胞高低转移能力株之间TRPM7表达量是否存在差异,发现差异后应用免疫细胞荧光技术检测TRPM7在阳性肿瘤细胞株中的表达部位和情况。选取该癌种的高转移细胞(高表达TRPM7)和低转移细胞(低表达TRPM7)分别进行后续的RNA干扰、基因转染及进一步的功能和机制实验研究。
2.瞬时干扰TRPM7对人鼻咽癌高转移细胞株5-8F迁移能力的影响
利用RNA干扰(RNAi)技术瞬时干扰高表达TRPM7的高转移细胞株5-8F的TRPM7 mRNA表达量,经RT-PCR、Western Blot检验TRPM7的干扰效果。体外分别用细胞划痕实验及transwell小室实验检测已成功干扰细胞的迁移能力变化。
3.稳定转染TRPM7对人鼻咽癌低转移细胞株6－10B迁移能力的影响
hTRPM7/pCDNA4/TO重组表达质粒由美国西雅图儿童医院Scharenberg实验室构建并作为礼物赠送我中心实验室,然后采用Invitrogen公司的Lipofectamine 2000TM分别将空质粒和TRPM7表达质粒hTRPM7/pCDNA4/TO转染进入6－10B细胞,用zeocin筛选阳性细胞克隆并扩增培养。并应用诱导剂Doxycycline诱导24小时后,应用RT-PCR与Western Blot检测稳定转染细胞的TRPM7基因及蛋白表达水平改变。分别用细胞划痕实验及transwell小室实验检测成功转染细胞的迁移能力变化。
4. TRPM7影响5-8F细胞迁移能力机制的初步探讨
利用荧光钙染料Fura-2并通过荧光分光光度计来检测细胞内钙变化情况,同时应用各种TRPM7离子通道特异性和非特异性的激活剂和阻断剂来探讨TRPM7表达水平对细胞内钙变化影响的机制以及内钙变化对细胞迁移的影响。
结果：
1.TRPM7基因和蛋白在5大瘤种10株转移能力不同肿瘤细胞株中的表达情况
采用RT-PCR法及Western blot检测收集到的5大瘤种10株转移能力不同肿瘤细胞株中TRPM7基因及蛋白的表达情况。结果表明：在5大瘤种的细胞株中,乳腺癌细胞株MCF-7与MDA-MB-231之间的TRPM7 mRNA (t=-2.545,P＝0.064)及蛋白(t=＝-2.554,P=0.063)表达量无显著性差异,肺癌细胞株Spca-1与A549之间的TRPM7 mRNA (t=1.466, P=0.217)及蛋白(t=-0.660,P=0.546)表达量无显著性差异,大肠癌细胞株SW480与SW620之间的TRPM7 mRNA(t=0.015,P=0.989)及蛋白(t=-2.609,P=0.059)表达量无显著性差异,肝癌细胞株HepG2与M6之间的TRPM7 mRNA(t=0.467, P=0.665)及蛋白(t=-2.642,P=0.057)表达量无显著性差异,而只有鼻咽癌细胞5－8F与6－10B之间的TRPM7 mRNA (t=-31.03, P<0.001)及蛋白(t=-59.937,P<0.001)表达量存在显著性差异。这一结果提示TRPM7可能为潜在的鼻咽癌转移相关因子。
2.TRPM7基因沉默对人鼻咽癌细胞5-8F迁移能力的影响
利用基因公司设计合成的3条针对TRPM7的siRNA片段及1条阴性对照siRNA片段,然后分别将这4条siRNA片段转染进入不同的5－8F细胞,利用RT-PCR和Western blot技术分别检测TRPM7的表达情况,筛选siRNA1-3干扰片段中干扰效率最高的siRNA1(80%)为下一步实验的研究对象(命名为5-8F/TRPM7细胞)。
细胞划痕实验检测TRPM7基因沉默后对高转移鼻咽癌细胞5-8F体外运动迁移能力的影响。实验结果显示：各组整体比较有显著性差异(F＝63.088,P<0.001),其中,5-8F/siRNA1、5-8F/siRNA2、5-8F/siRNA3细胞迁移能力显著低于5-8F／NC,差异具有显著性(S1－3均是P<0.001),表明降低TRPM7表达后5－8F细胞迁移能力显著减弱。
同样,体外transwell小室实验检测TRPM7基因表达沉默后5-8F细胞迁移能力的改变。结果显示,各组整体比较有显著性差异(F=165.098,P<0.001)。其中,与5-8F细胞和5-8F/empty细胞相比,5-8F/TRPM7细胞的迁移能力显著降低(S1－3均是P<0.001),说明干扰TRPM7基因显著抑制了鼻咽癌细胞的迁移能力。3. TRPM7基因过表达对人鼻咽癌细胞6－10B迁移能力的影响
分别将空质粒和TRPM7表达质粒转染进入原本低表达TRPM7的6－10B细胞,用zeocin筛选阳性细胞克隆并扩增培养。应用DOX诱导24小时,然后利用RT-PCR和Western blot分别检测TRPM7的表达情况,筛选转染表达效率最高的细胞作为下一步实验的研究对象(命名为6-10B/TRPM7+细胞)。
细胞划痕实验检测TRPM7基因过表达后对低转移鼻咽癌细胞6－10B体外运动迁移能力的影响。实验结果显示：各组整体比较有显著性差异(F=65.592,P<0.001)。其中,6-10B/TRPM7+细胞迁移数明显高于6－10B和6-10B/empty,差异具有显著性(各组均是P<0.001)。表明6-10B/TRPM7+细胞的运动迁移能力要强于6－10B细胞和6-10B/empty细胞。
同样,体外transwell小室实验检测TRPM7基因过表达后细胞迁移能力的改变,结果显示,各组整体比较有显著性差异(F=300.00,P<0.001)。其中,与6－10B细胞和6-10B/empty细胞相比,6-10B/TRPM7+细胞的迁移能力显著提高(各组均是P<0.001),说明TRPM7基因的过表达增强了鼻咽癌细胞的迁移能力。
4. TRPM7的表达高低对鼻咽癌细胞5-8F内钙变化和细胞迁移影响的初步机制探讨
由于TRPM7是一种细胞膜表面的钙离子通道,所以我们应用药理学结合划痕实验等方法来鉴定细胞外的钙离子内流对于5-8F细胞迁移的影响。胞外钙离子螯合剂EGTA作用后显著抑制了该细胞的迁移(P<0.001),这一结果说明外钙的内流是5-8F细胞迁移的前提；由于细胞膜上有可能存在各种各样的钙离子通道如电压门控式、受体依赖式、钙库调控式或是我们的研究对象瞬时受体电位通道即TRP家族,因此,我们使用上述各种相应的离子通道阻断剂来验证究竟是何种通道在主要起介导胞外钙内流的作用。结果显示：除外TRP通道的上述其他3种通道阻断剂,SKF96365：钙库调控钙通道阻断剂(P＝0.968)：Nimodipine:电压门控式钙通道阻断剂(P=0.687); 2-AP:NMDA受体阻断剂(P=1.000); CNQX:AMPA受体阻断剂(P=0.928)与对照组相比均未能对5－8F细胞的迁移造成显著的影响,而TRPM7的非特异性阻断剂La3+(P<0.001)和2-APB(P<0.001)则显著的抑制了5-8F的迁移能力；相反,应用TRPM7特异的激活剂BK却显著提高了5-8F细胞的迁移能力(P=0.038)。综上结果,我们可以初步确定TRP通道是5-8F细胞外钙内流的主要调控通道,而结合前面的TRPM7干扰和转染实验,我们可以得出结论：TRPM7主要介导了外钙的内流并成为TRP家族中调控鼻咽癌细胞迁移的主要分子。
接下来,我们进一步应用钙染料Fura-2/AM来检测5-8F细胞内的钙变化,以便进一步探讨TRPM7对5-8F细胞内钙离子变化的调控机制。由TRPM7激活剂BK诱导的胞内钙变化主要包含一个来自胞内钙库钙离子迅速释放而形成的快升快降的钙高峰期和一个主要由TRPM7介导的持续数分钟的缓慢下降的外钙内流平台期。
利用我们前面已经提到的瞬时RNA干扰技术,我们选用钙染料Fura-2/AM来观察TRPM7干扰前后对5-8F胞内钙浓度造成的影响。结果提示：TRPM7明显的降低了平台期的胞内钙浓度,而对高峰期的钙反应几乎没有影响。说明平台期钙内流主要由TRPM7介导。
TRPM7非特异性阻断剂La3+和2-APB几乎完全抑制包括高峰期和平台期在内的所有5-8F细胞内的钙变化,应用胞外钙离子螯合剂EGTA同样可以观察到相同的结果；这些数据表明：在没有外钙存在和介导外钙内流的钙离子通道存在的前提下,5-8F细胞不会有任何钙变化,由此我们也可以确定,胞内钙库释放主要由胞外钙内流介导,即可能是通过比较少见的钙诱导的钙库释放机制(CICR)。
此外,为了进一步明确外钙内流与胞内钙库释放之间的关系和具体调控机理,我们分别应用ryanodine(斯里兰卡肉桂碱,RyR)受体阻断剂ryanodine和IP3(1,4,5-三磷酸肌醇)受体阻断剂xestospongin C分别来阻断由BK诱导的“TRPM7样”钙反应。结果提示：IP3R阻断剂ryanodine对BK诱生的胞内钙库释放和外钙内流均没有任何影响,而RyR阻断剂ryanodine则几乎抑制了高峰期的钙变化即抑制了胞内钙库的释放,但对平台期TRPM7介导的外钙缓慢内流没有影响。这些结果提示：TRPM7钙离子通道主要通过介导外钙内流从而通过CICR机制激活胞内钙库释放从而影响整个胞内钙反应。
结论
1.TRPM7基因／蛋白在鼻咽癌高转移细胞株5-8F中高表达而在低转移株6－10B中低表达,表明TRPM7可能是促进鼻咽癌转移的潜在转移相关分子和预后因子。
2.TRPM7的表达高低决定了鼻咽癌细胞迁移能力的高低,提示TRPM7是鼻咽癌细胞的迁移相关分子。
3.TRPM7是胞内钙变化的主要启动子和调控子,提示TRPM7很可能成为鼻咽癌患者治疗的靶点。
本研究的创新之处
1.首次发现TRPM7与肿瘤转移(鼻咽癌细胞迁移)相关,为开展鼻咽癌基因靶向治疗初步奠定了理论基础；
2.初步明确了TRPM7是鼻咽癌胞内钙变化的新的启动和调控分子,这对于研究控制鼻咽癌转移提供了一个潜在治疗和预后指标；
3.首次阐明了TRPM7如何影响鼻咽癌细胞内钙变化从而影响细胞迁移以及肿瘤细胞内存在由TRPM7介导的外钙内流激活的钙诱导钙库释放(CICR)机制。
4.利用质粒转染技术建立了TRPM7基因过表达的稳定鼻咽癌细胞株6-10B/TRPM7+,为研究TRPM7基因的功能提供了有价值的研究工具。
Background and Objective:
Although nasopharyngeal carcinoma (NPC) is rare in Western countries, it is extremely common in southern regions of China. In Guangdong, for example, NPC accounts for 18% of all cancers. Because NPC occurs close to the intracranial organs, the cancer cells invade the cranial cavity and metastasize. This leads to a poor prognosis for many NPC patients. Therefore, an improved understanding of the molecular mechanisms involved in the intracranial invasion and metastasis of NPC will lead to improved treatments and prognosis factors for NPC patients.
Cell migration is required during the invasion and metastasis of tumor cells, and the ubiquitous second messenger Ca2+ is a critical regulator of cell migration. Recently, many studies have shown that Ca2+ influx and Ca2+ channels are essential for the migration of various cell types, including tumor cells such as breast cancer cells. It is possible, therefore, that Ca2+ entry pathways also exist in NPC cells. Tumor cells often lack the voltage-operated Ca2+ channels and receptor-operated Ca2+ channels that play a pivotal role in Ca2+ entry in excitable cells. However, recent studies of tumor cells have revealed two other potential pathways for Ca2+ entry:store-operated Ca2+ channels and transient receptor potential (TRP) channels. Three TRP channels, TRPM1, TRPM8 and TRPV6, have been shown to control Ca influx, and to regulate the migration of murine melanoma cells (B16-F1/10), human glioblastoma cells (DBTRG) and hepatoblastoma (HepG2) cells, respectively.
TRP channels were first identified in Drosophila species. On the bases of homology and channel function, the TRP family is divided into three main subfamilies:classic (TRPC), vanilloid (TRPV), and melastatin (TRPM). TRPM7, a member of the TRPM subfamily, is a non-selective cation channel with predominant permeability for Ca2+ and Mg2+. It regulates the calcium concentration in cells, which is imperative for many processes. Recent studies have suggested more and more importance for TRPM7 in the processes of cell migration. Wei showed that calcium flickers, which arose from TRPM7 at the migrating fibroblast front, had a crucial role in guiding directional movement—after TRPM7 knockdown and inhibition of calcium signalling, all migratory, turning, and chemotactic abilities were impaired. Clark demonstrated that activating TRPM7 could promote cytoskeletal relaxation and the conversion of focal adhesions to podosomes in mouse tumor cells (N1E-115). Despite these findings, the potential function of TRPM7 channels in the migration of tumor cells is not known. We hypothesized that TRPM7 Ca2+ channels are important in the migration of human NPC cells.
We used colony lines of the NPC SUNE1 cell line to investigate the presence of TRPM7 channels in 5-8F cells (highly tumorigenic with metastatic ability) and 6-10B cells (tumorigenic without metastatic ability).We found high expression of the TRPM7 gene and protein in 5-8F cells, but low expression in 6-10B cells. By activation, blockade, and knockdown of these ion channels, we showed that TRPM7 affected the migratory potential of 5-8F cells. Contrary to TRPM7 RNAi results in 5-8F cells, we found that overexpression of TRPM7 increased the migration of 6-10B cells. Moreover, we found that TRPM7 regulated the migration of 5-8F cells by controlling Ca2+ influx. Finally, pharmacological data showed that TRPM7 channels were not stores-operated channels but, instead, controlled the release of intracellular Ca2+ stores via ryanodine receptors by a calcium-induced calcium release (CICR) mechanism. Overall, our data suggest that TRPM7 might be critical for the migration of NPC cells.
Methods:
1. Cell and Cell culture
Two colony lines of human NPC SUNE1 cells—5-8F cells, which are highly tumorigenic and have metastatic ability, and 6-10B cells, which are tumorigenic, but lack metastatic ability—were stored in our laboratory. Both cell lines originated from a poorly differentiated squamous cell carcinoma of the nasopharynx.
2. Small interfering RNA silencing
All siRNA duplexes were synthesized by Shanghai GenePharma Co. In the wells, three TRPM7 siRNA or negative control siRNA, and Lipofect AMINE 2000 was added to Opti-MEM and mixed gently. The wells were added to plates and incubated for 48 h until ready for further assay. All western blotting and functional studies were carried out after a 48 h-72 h transfection.
3. Construct and 6-10B cells transfection
The expression construct WT human TRPM7/pCDNA4/TO were gifts from Scharenberg Lab. This recombinant hTRPM7 is tagged with the hemagglutinin epitope.6-10B cells were transfected with the hTRPM7/pCDNA4/TO construct and empty construct by using Lipofectamine 2000 and selected for stable transfectants by zeocin treatment. TRPM7 expression was induced 1 day before use by adding 1μg/ml doxycycline to the culture medium.6-10B cells transfected with the empty vector served as a control for all experiments. Western blot analysis, wound healing assay and transwell chamber migration assay were performed 16-24 h after induction.
4. Calcium imaging
Calcium-imaging experiments were performed as described previously. Briefly, cells were plated onto glass coverslips and loaded with Fura-2 AM. Cells were then placed in a perfusion chamber on the stage of the microscope with a 40×objective. Fluorescence images of the cells were recorded by a Ratio Vision digital fluorescence microscopy system and analysed by TILL vision 4.0 software.
5. Wound healing assay
5-8F cells were cultured in 12 well tissue culture plates to confluence without vacant space. A pipette tip was used to scratch a wound midline of the culture well then cells were pretreated with mitomycin C. After 16 h of culture in RPMI 1640 supplemented with 2% serum, migration of cells was evaluated by measuring the difference in width of the wounds at 0 h and at 16 h.
6. Transwell chamber migration assay
The migration activities of 5-8F cells, siNC-transfected 5-8F cells, and siTRPM7-transfected 5-8F cells were assayed using transwell cell culture chambers. Cells were added to the transwell chamber. The motilities of NC and TRPM7 knockdown cells in the transwell chamber assay were stimulated by the serum alone or the serum in combination with BK. The serum in bottom wells acts as chemotatic factors and BK in both top and bottom wells acts as expected motility boosting factors. After 48 hours the cells that migrated through the membrane were counted. Cells were counted in 5 random fields and expressed as the average number of cells per field under a light microscope.
7. Statistical analysis
Statistical data are expressed as mean±SEM. When appropriate, Student's t-test and one-way ANOVA were applied. In all cases, p<0.05 was considered statistically significant. All experiments were repeated at least three times.
Results:
1. Expression of TRPM7 higher in 5-8F cells than in 6-10B cells The expression of TRPM7 in 5-8F and 6-10B cells was examined by western blot, RT-PCR and immunofluorescence staining. Levels of TRPM7—both mRNA and protein—were significantly higher in 5-8F cells than in 6-10B cells.
2. Silencing of TRPM7 inhibits migration of 5-8F cells Immunoblotting analyses showed that, within 48 h after transfection of 5-8F cells with TRPM7 siRNA No.1-3, the expression of TRPM7 protein was suppressed 65%-80%. Moreover, wound-healing assays showed that serum-induced migration of 5-8F cells was inhibited 50%-70%by TRPM7 siRNA. Finally, the motility of TRPM7 knockdown cells was analyzed using the transwell chamber assay. Like the down-regulated expression of TRPM7 and the decreased influx of Ca2+, motility was also reduced in cells transfected with TRPM7 siRNA. Compared with negative control siRNA cells, TRPM7 knockdown (siRNA1) cells showed a 68%reduction in the number of cells that crossed the membrane, indicating that TRPM7 channels were required for migration.
3. Overexpression of TRPM7 enhances migration of 6-10 cells Immunoblotting analyses showed that, within 48 h after transfection of 6-10B cells with hTRPM7/pCDNA4/TO, the expression of TRPM7 protein was improved significantly.Wound-healing assays and transwell chamber assays showed that BK and serum in combination or serum-induced migratory abilities increased 260%-460%in 6-10B cells transfected with WT-hTRPM7 construct when compared with control 6-10B cells.
4. Ca2+ influx mediated by TRPM7 potentially critical for migration of 5-8F cells Pharmacological approaches were used to verify that influx of extracellular Ca2+ affected the migration of 5-8F cells. In the wound-healing assay, removal of extracellular Ca2+ by EGTA significantly inhibited migration of 5-8F cells, indicating that influx of extracellular Ca2+ is critical for the migration of 5-8F cells. Next, wound-healing assays were employed to investigate the effect on cell migration of pharmacological inhibitors for various ion channels that mediated Ca2+ influx. Results showed that four inhibitors of three different Ca2+ channels had no effect on migration of 5-8F cells. However, the addition of a non-specific TRPM7 channel inhibitor 2-APB reduced the migration of 5-8F cells. Likewise, another non-specific TRPM7 channel inhibitor La3+ inhibited the migration of 5-8F cells. Conversely, the addition of BK, a peptide agonist that activates and promotes expression of TRPM7 in N1E-115 cells, promoted the migration of 5-8F cells. Transwell chamber assays also revealed that both 2-APB and La3+ inhibited the migration of 5-8F cells.
5. TRPM7 channel potentially a major regulator of intracellular Ca2+ response In pharmacological approaches to further confirm that TRPM7 mediated Ca2+ influx in 5-8F cells, Fura-2-based Ca2+ imaging was performed. Addition of BK triggered a rapid increase in cytosolic Ca2+ from internal stores in 5-8F cells. The initial increase in Ca2+ was transient, but was followed by a sustained phase of elevated Ca2+ that lasted for several minutes before Ca2+ returned to basal levels. Ratiometric analysis showed that compared with control (NC), silencing of TRPM7 (siRNA1) significantly decreased the transient phase of the response, but it decreased the sustained phase of the response much greater.
Fura-2-based Ca2+ imaging was also used to examine the mechanism by which TRPM7 inhibitors influenced influx of extracellular Ca2+ and release of intracellular Ca2+ stores in 5-8F cells. Both of TRPM7-mediated Ca2+ influx and Ca2+ store Ca2+ release could be induced consecutively two times by BK without any desensitization in the control group. However, compared with the control group, both La3+ and 2-APB completely inhibited the amplitude of the peak and plateau phases of the second BK-induced Ca2+ response. Interestingly, similar results were observed with EGTA, which chelated extracellular Ca2+. These data indicated that Ca2+ response was not induced by BK without extracellular Ca2+.
Moreover, ryanodine receptor inhibitor ryanodine and inositol-1,4,5-trisphosphate receptor inhibitor xestospongin C were used to investigate whether calcium-induced calcium release occurred via the RyR or IP3R in 5-8F cells. We compared the first response to BK in the absence of ryanodine with the corresponding second BK response after ryanodine pre-incubation. These results showed that ryanodine significantly reduced BK-induced Ca2+ peak responses while having no significant effect on BK-induced Ca2+ plateau phases. However, different results were obtained by inhibiting the IP3R with xestospongin C. The corresponding second BK response after xestospongin C pre-incubation was similar with the first BK response.
Taken together, these results in 5-8F cells confirm and characterize the mediating effects of TRPM7 channels, both on influx of extracellular Ca2+ and release of intracellular Ca2+ stores via RyRs, but not IP3Rs.
Conclusions:
This study supports the influx of Ca2+ as a requirement for the migration of human NPC 5-8F cells and identifies TRPM7 as a novel regulator not only of Ca2+ influx but also of 5-8F cell migration, thus showing TRPM7 as a potential prognostic indicator and therapeutic target for NPC patients.
Innovations of our study:
1. We were the first to confirm how TRPM7 channels function to regulate the migration of tumor cells.
2. Our preliminary study demonstrated TRPM7 was a key gene/protein related to migration of NPC.
3. Our study identified TRPM7 as a novel potential-regulator of the Ca2+ influx that allows migration of 5-8F cells. TRPM7, therefore, might have potential as a prognostic indicator and as a therapeutic target in nasopharyngeal carcinoma.
4. Stable TRPM7-transduced nasopharyngeal carcinoma cell line 6-10B was established, which will be provided a valuable tool for TRPM7 functional studies.
目前恶性肿瘤的治疗之所以失败的原因,一是耐药性,二是无法防治肿瘤的转移。恶性肿瘤在诊断时局部淋巴结转移发生率为1／3－1／4。而肿瘤致死亡的主要原因就是肿瘤细胞的转移,90%肿瘤患者最终死于转移。但当前人们对肿瘤转移机制缺乏系统而深入的认识；现行临床治疗手段对抑制肿瘤转移基本无效；故寻找肿瘤转移相关因子,揭示肿瘤转移的分子机制便成为当今肿瘤研究面临的最大挑战及国际研究的热点。
肿瘤细胞的侵袭转移涉及肿瘤细胞及间质细胞和基质之间的多个步骤和分子反应。目前肿瘤转移步骤的模式广为人们接受的是Liotta提出的癌细胞侵袭转移的三步骤假说：1.肿瘤细胞之间的解黏附和肿瘤细胞与ECM之间的黏附；2.侵足的形成与ECM的降解：肿瘤细胞和宿主细胞分泌的蛋白水解酶,使肿瘤细胞周围的发生降解；3.运动,肿瘤细胞被生长因子及趋化因子诱导,向纵深运动。值得一提的是,虽然上述个阶段涉及到相对特异的分子机制,但是侵袭转移过程中各阶段之间的分子存在交叉和相互作用,因此侵袭转移的阶段和分子功能划分只是相对的。
已有研究证明,正常组织细胞在特殊情况下也具备迁移能力,如胚胎发育、创伤愈合、免疫细胞。而肿瘤细胞由正常细胞经过演变,形成强于正常组织细胞的转移能力。肿瘤细胞虽然具备自身特点,但由于来源于正常组织细胞,因此在很多方面还是原有功能的沿用或是增强。而当今一些肿瘤转移重大发现采用了类比的方式,跨学科从生理学中寻找对于正常细胞迁移的重要分子,试图找到对于肿瘤细胞至关重要的转移相关因子。比如肿瘤生物学最新研究表明：在胚胎发育中起重要作用的上皮至间质转换(Epithelial-mesenchymal transition, EMT)便是肿瘤转移的重要机制之一。我们的研究思路正是采用这种交叉学科间的类比思维,试图通过寻找在正常细胞中证实已与细胞迁移密切相关的因子,同时收集5大瘤种10株转移能力不同的肿瘤细胞,从基因和蛋白水平比较,而非采取常规的高通量基因蛋白芯片,试图找到与肿瘤细胞转移能力相关的分子而进一步对其进行深入研究。经过文献调研,我们认为瞬时受体电位通道家族之一的TRPM7可能与肿瘤细胞的迁移相关,初步证据如下：
瞬时受体电位通道(TRP channels)是位于细胞膜上的一类重要的阳离子通道超家族。TRP通道分为：TRPC、TRPV、TRPM、TRPA、TRPP和TRPML6大亚家族。TRPM (Melastatin)亚家族包括TRPM1-8,共8种亚型。TRP通道为非选择性阳离子通道,TRPM7通道主要通过的离子为钙离子、钠离子,其还可通过镁、锌、锰等很多二价离子。
北京大学程和平等运用共聚焦显微成像技术,发现迁移成纤维细胞头部存在大量微小而短暂的钙信号事件,即“钙闪烁”现象,并证明钙闪烁起着掌控细胞运动“方向舵”的作用。实验条件下,钙闪烁被抑制的细胞可以维持直线运动,但完全丧失了定向和转弯的能力。进而提出了“钙闪烁引导细胞定向迁移”的新观点：在外界趋化因子梯度诱导下,钙闪烁发放呈现不对称特性,即趋化因子浓度高的一侧,钙闪烁更为活跃,驱动细胞转向此侧,从而精确地调控细胞的定向迁移。而在文章中作者通过实验验证：在所有可能的TRP通道中,TRPM7通道是唯一调控迁移细胞前端的“钙闪烁”现象的通道蛋白,即TRPM7才是真正掌控细胞运动的“方向舵”,并以此来指引细胞跟随趋化因子梯度进而有目的的定向迁移。
此外,Kristopher Clark等发现在小鼠N1E-115神经母细胞瘤细胞中,TRPM7介导缓激肽(BK)诱导的钙内流；同时过表达TRPM7可以促进该细胞的伸展并增强细胞与基质之间的粘附；此外TRPM7的激活还可以导致激酶依赖的足体的形成。而对于足体,目前研究表明：正常细胞的足体与肿瘤细胞的侵足关系密切,人们推测足体是侵入性伪足的前体,在一定条件下形成稳定的侵入性伪足。足体在生理物质上显示出降解细胞外基质的功能。我们也可以由此推测：TRPM7也可能在肿瘤细胞中通过导致侵足的形成继而增强肿瘤细胞的侵袭能力。另外,还有研究表明：TRPM7可与细胞膜表面的马达蛋白myosinⅡ相互作用,从而通过调控细胞骨架解除细胞与基质的粘附进而影响细胞的伸展。
综上,TRPM7已被证明的一系列细胞迁移相关的作用与肿瘤细胞迁移、侵袭的各个阶段均具有相关性,故我们大胆推测：TRPM7可能是肿瘤细胞侵袭转移的重要调控分子。
方法：
1.肿瘤细胞株的选取及TRPM7基因／蛋白水平表达的差异检测
选取分属鼻咽癌、乳腺癌、肺癌、大肠癌、肝癌等5大瘤种中已公认转移能力不同的10株肿瘤细胞株为研究对比对象。运用RT-PCR和Western Blot从基因和蛋白两个表达水平检测不同瘤种的肿瘤细胞高低转移能力株之间TRPM7表达量是否存在差异,发现差异后应用免疫细胞荧光技术检测TRPM7在阳性肿瘤细胞株中的表达部位和情况。选取该癌种的高转移细胞(高表达TRPM7)和低转移细胞(低表达TRPM7)分别进行后续的RNA干扰、基因转染及进一步的功能和机制实验研究。
2.瞬时干扰TRPM7对人鼻咽癌高转移细胞株5-8F迁移能力的影响
利用RNA干扰(RNAi)技术瞬时干扰高表达TRPM7的高转移细胞株5-8F的TRPM7 mRNA表达量,经RT-PCR、Western Blot检验TRPM7的干扰效果。体外分别用细胞划痕实验及transwell小室实验检测已成功干扰细胞的迁移能力变化。
3.稳定转染TRPM7对人鼻咽癌低转移细胞株6－10B迁移能力的影响
hTRPM7/pCDNA4/TO重组表达质粒由美国西雅图儿童医院Scharenberg实验室构建并作为礼物赠送我中心实验室,然后采用Invitrogen公司的Lipofectamine 2000TM分别将空质粒和TRPM7表达质粒hTRPM7/pCDNA4/TO转染进入6－10B细胞,用zeocin筛选阳性细胞克隆并扩增培养。并应用诱导剂Doxycycline诱导24小时后,应用RT-PCR与Western Blot检测稳定转染细胞的TRPM7基因及蛋白表达水平改变。分别用细胞划痕实验及transwell小室实验检测成功转染细胞的迁移能力变化。
4. TRPM7影响5-8F细胞迁移能力机制的初步探讨
利用荧光钙染料Fura-2并通过荧光分光光度计来检测细胞内钙变化情况,同时应用各种TRPM7离子通道特异性和非特异性的激活剂和阻断剂来探讨TRPM7表达水平对细胞内钙变化影响的机制以及内钙变化对细胞迁移的影响。
结果：
1.TRPM7基因和蛋白在5大瘤种10株转移能力不同肿瘤细胞株中的表达情况
采用RT-PCR法及Western blot检测收集到的5大瘤种10株转移能力不同肿瘤细胞株中TRPM7基因及蛋白的表达情况。结果表明：在5大瘤种的细胞株中,乳腺癌细胞株MCF-7与MDA-MB-231之间的TRPM7 mRNA (t=-2.545,P＝0.064)及蛋白(t=＝-2.554,P=0.063)表达量无显著性差异,肺癌细胞株Spca-1与A549之间的TRPM7 mRNA (t=1.466, P=0.217)及蛋白(t=-0.660,P=0.546)表达量无显著性差异,大肠癌细胞株SW480与SW620之间的TRPM7 mRNA(t=0.015,P=0.989)及蛋白(t=-2.609,P=0.059)表达量无显著性差异,肝癌细胞株HepG2与M6之间的TRPM7 mRNA(t=0.467, P=0.665)及蛋白(t=-2.642,P=0.057)表达量无显著性差异,而只有鼻咽癌细胞5－8F与6－10B之间的TRPM7 mRNA (t=-31.03, P<0.001)及蛋白(t=-59.937,P<0.001)表达量存在显著性差异。这一结果提示TRPM7可能为潜在的鼻咽癌转移相关因子。
2.TRPM7基因沉默对人鼻咽癌细胞5-8F迁移能力的影响
利用基因公司设计合成的3条针对TRPM7的siRNA片段及1条阴性对照siRNA片段,然后分别将这4条siRNA片段转染进入不同的5－8F细胞,利用RT-PCR和Western blot技术分别检测TRPM7的表达情况,筛选siRNA1-3干扰片段中干扰效率最高的siRNA1(80%)为下一步实验的研究对象(命名为5-8F/TRPM7细胞)。
细胞划痕实验检测TRPM7基因沉默后对高转移鼻咽癌细胞5-8F体外运动迁移能力的影响。实验结果显示：各组整体比较有显著性差异(F＝63.088,P<0.001),其中,5-8F/siRNA1、5-8F/siRNA2、5-8F/siRNA3细胞迁移能力显著低于5-8F／NC,差异具有显著性(S1－3均是P<0.001),表明降低TRPM7表达后5－8F细胞迁移能力显著减弱。
同样,体外transwell小室实验检测TRPM7基因表达沉默后5-8F细胞迁移能力的改变。结果显示,各组整体比较有显著性差异(F=165.098,P<0.001)。其中,与5-8F细胞和5-8F/empty细胞相比,5-8F/TRPM7细胞的迁移能力显著降低(S1－3均是P<0.001),说明干扰TRPM7基因显著抑制了鼻咽癌细胞的迁移能力。3. TRPM7基因过表达对人鼻咽癌细胞6－10B迁移能力的影响
分别将空质粒和TRPM7表达质粒转染进入原本低表达TRPM7的6－10B细胞,用zeocin筛选阳性细胞克隆并扩增培养。应用DOX诱导24小时,然后利用RT-PCR和Western blot分别检测TRPM7的表达情况,筛选转染表达效率最高的细胞作为下一步实验的研究对象(命名为6-10B/TRPM7+细胞)。
细胞划痕实验检测TRPM7基因过表达后对低转移鼻咽癌细胞6－10B体外运动迁移能力的影响。实验结果显示：各组整体比较有显著性差异(F=65.592,P<0.001)。其中,6-10B/TRPM7+细胞迁移数明显高于6－10B和6-10B/empty,差异具有显著性(各组均是P<0.001)。表明6-10B/TRPM7+细胞的运动迁移能力要强于6－10B细胞和6-10B/empty细胞。
同样,体外transwell小室实验检测TRPM7基因过表达后细胞迁移能力的改变,结果显示,各组整体比较有显著性差异(F=300.00,P<0.001)。其中,与6－10B细胞和6-10B/empty细胞相比,6-10B/TRPM7+细胞的迁移能力显著提高(各组均是P<0.001),说明TRPM7基因的过表达增强了鼻咽癌细胞的迁移能力。
4. TRPM7的表达高低对鼻咽癌细胞5-8F内钙变化和细胞迁移影响的初步机制探讨
由于TRPM7是一种细胞膜表面的钙离子通道,所以我们应用药理学结合划痕实验等方法来鉴定细胞外的钙离子内流对于5-8F细胞迁移的影响。胞外钙离子螯合剂EGTA作用后显著抑制了该细胞的迁移(P<0.001),这一结果说明外钙的内流是5-8F细胞迁移的前提；由于细胞膜上有可能存在各种各样的钙离子通道如电压门控式、受体依赖式、钙库调控式或是我们的研究对象瞬时受体电位通道即TRP家族,因此,我们使用上述各种相应的离子通道阻断剂来验证究竟是何种通道在主要起介导胞外钙内流的作用。结果显示：除外TRP通道的上述其他3种通道阻断剂,SKF96365：钙库调控钙通道阻断剂(P＝0.968)：Nimodipine:电压门控式钙通道阻断剂(P=0.687); 2-AP:NMDA受体阻断剂(P=1.000); CNQX:AMPA受体阻断剂(P=0.928)与对照组相比均未能对5－8F细胞的迁移造成显著的影响,而TRPM7的非特异性阻断剂La3+(P<0.001)和2-APB(P<0.001)则显著的抑制了5-8F的迁移能力；相反,应用TRPM7特异的激活剂BK却显著提高了5-8F细胞的迁移能力(P=0.038)。综上结果,我们可以初步确定TRP通道是5-8F细胞外钙内流的主要调控通道,而结合前面的TRPM7干扰和转染实验,我们可以得出结论：TRPM7主要介导了外钙的内流并成为TRP家族中调控鼻咽癌细胞迁移的主要分子。
接下来,我们进一步应用钙染料Fura-2/AM来检测5-8F细胞内的钙变化,以便进一步探讨TRPM7对5-8F细胞内钙离子变化的调控机制。由TRPM7激活剂BK诱导的胞内钙变化主要包含一个来自胞内钙库钙离子迅速释放而形成的快升快降的钙高峰期和一个主要由TRPM7介导的持续数分钟的缓慢下降的外钙内流平台期。
利用我们前面已经提到的瞬时RNA干扰技术,我们选用钙染料Fura-2/AM来观察TRPM7干扰前后对5-8F胞内钙浓度造成的影响。结果提示：TRPM7明显的降低了平台期的胞内钙浓度,而对高峰期的钙反应几乎没有影响。说明平台期钙内流主要由TRPM7介导。
TRPM7非特异性阻断剂La3+和2-APB几乎完全抑制包括高峰期和平台期在内的所有5-8F细胞内的钙变化,应用胞外钙离子螯合剂EGTA同样可以观察到相同的结果；这些数据表明：在没有外钙存在和介导外钙内流的钙离子通道存在的前提下,5-8F细胞不会有任何钙变化,由此我们也可以确定,胞内钙库释放主要由胞外钙内流介导,即可能是通过比较少见的钙诱导的钙库释放机制(CICR)。
此外,为了进一步明确外钙内流与胞内钙库释放之间的关系和具体调控机理,我们分别应用ryanodine(斯里兰卡肉桂碱,RyR)受体阻断剂ryanodine和IP3(1,4,5-三磷酸肌醇)受体阻断剂xestospongin C分别来阻断由BK诱导的“TRPM7样”钙反应。结果提示：IP3R阻断剂ryanodine对BK诱生的胞内钙库释放和外钙内流均没有任何影响,而RyR阻断剂ryanodine则几乎抑制了高峰期的钙变化即抑制了胞内钙库的释放,但对平台期TRPM7介导的外钙缓慢内流没有影响。这些结果提示：TRPM7钙离子通道主要通过介导外钙内流从而通过CICR机制激活胞内钙库释放从而影响整个胞内钙反应。
结论
1.TRPM7基因／蛋白在鼻咽癌高转移细胞株5-8F中高表达而在低转移株6－10B中低表达,表明TRPM7可能是促进鼻咽癌转移的潜在转移相关分子和预后因子。
2.TRPM7的表达高低决定了鼻咽癌细胞迁移能力的高低,提示TRPM7是鼻咽癌细胞的迁移相关分子。
3.TRPM7是胞内钙变化的主要启动子和调控子,提示TRPM7很可能成为鼻咽癌患者治疗的靶点。
本研究的创新之处
1.首次发现TRPM7与肿瘤转移(鼻咽癌细胞迁移)相关,为开展鼻咽癌基因靶向治疗初步奠定了理论基础；
2.初步明确了TRPM7是鼻咽癌胞内钙变化的新的启动和调控分子,这对于研究控制鼻咽癌转移提供了一个潜在治疗和预后指标；
3.首次阐明了TRPM7如何影响鼻咽癌细胞内钙变化从而影响细胞迁移以及肿瘤细胞内存在由TRPM7介导的外钙内流激活的钙诱导钙库释放(CICR)机制。
4.利用质粒转染技术建立了TRPM7基因过表达的稳定鼻咽癌细胞株6-10B/TRPM7+,为研究TRPM7基因的功能提供了有价值的研究工具。
Background and Objective:
Although nasopharyngeal carcinoma (NPC) is rare in Western countries, it is extremely common in southern regions of China. In Guangdong, for example, NPC accounts for 18% of all cancers. Because NPC occurs close to the intracranial organs, the cancer cells invade the cranial cavity and metastasize. This leads to a poor prognosis for many NPC patients. Therefore, an improved understanding of the molecular mechanisms involved in the intracranial invasion and metastasis of NPC will lead to improved treatments and prognosis factors for NPC patients.
Cell migration is required during the invasion and metastasis of tumor cells, and the ubiquitous second messenger Ca2+ is a critical regulator of cell migration. Recently, many studies have shown that Ca2+ influx and Ca2+ channels are essential for the migration of various cell types, including tumor cells such as breast cancer cells. It is possible, therefore, that Ca2+ entry pathways also exist in NPC cells. Tumor cells often lack the voltage-operated Ca2+ channels and receptor-operated Ca2+ channels that play a pivotal role in Ca2+ entry in excitable cells. However, recent studies of tumor cells have revealed two other potential pathways for Ca2+ entry:store-operated Ca2+ channels and transient receptor potential (TRP) channels. Three TRP channels, TRPM1, TRPM8 and TRPV6, have been shown to control Ca influx, and to regulate the migration of murine melanoma cells (B16-F1/10), human glioblastoma cells (DBTRG) and hepatoblastoma (HepG2) cells, respectively.
TRP channels were first identified in Drosophila species. On the bases of homology and channel function, the TRP family is divided into three main subfamilies:classic (TRPC), vanilloid (TRPV), and melastatin (TRPM). TRPM7, a member of the TRPM subfamily, is a non-selective cation channel with predominant permeability for Ca2+ and Mg2+. It regulates the calcium concentration in cells, which is imperative for many processes. Recent studies have suggested more and more importance for TRPM7 in the processes of cell migration. Wei showed that calcium flickers, which arose from TRPM7 at the migrating fibroblast front, had a crucial role in guiding directional movement—after TRPM7 knockdown and inhibition of calcium signalling, all migratory, turning, and chemotactic abilities were impaired. Clark demonstrated that activating TRPM7 could promote cytoskeletal relaxation and the conversion of focal adhesions to podosomes in mouse tumor cells (N1E-115). Despite these findings, the potential function of TRPM7 channels in the migration of tumor cells is not known. We hypothesized that TRPM7 Ca2+ channels are important in the migration of human NPC cells.
We used colony lines of the NPC SUNE1 cell line to investigate the presence of TRPM7 channels in 5-8F cells (highly tumorigenic with metastatic ability) and 6-10B cells (tumorigenic without metastatic ability).We found high expression of the TRPM7 gene and protein in 5-8F cells, but low expression in 6-10B cells. By activation, blockade, and knockdown of these ion channels, we showed that TRPM7 affected the migratory potential of 5-8F cells. Contrary to TRPM7 RNAi results in 5-8F cells, we found that overexpression of TRPM7 increased the migration of 6-10B cells. Moreover, we found that TRPM7 regulated the migration of 5-8F cells by controlling Ca2+ influx. Finally, pharmacological data showed that TRPM7 channels were not stores-operated channels but, instead, controlled the release of intracellular Ca2+ stores via ryanodine receptors by a calcium-induced calcium release (CICR) mechanism. Overall, our data suggest that TRPM7 might be critical for the migration of NPC cells.
Methods:
1. Cell and Cell culture
Two colony lines of human NPC SUNE1 cells—5-8F cells, which are highly tumorigenic and have metastatic ability, and 6-10B cells, which are tumorigenic, but lack metastatic ability—were stored in our laboratory. Both cell lines originated from a poorly differentiated squamous cell carcinoma of the nasopharynx.
2. Small interfering RNA silencing
All siRNA duplexes were synthesized by Shanghai GenePharma Co. In the wells, three TRPM7 siRNA or negative control siRNA, and Lipofect AMINE 2000 was added to Opti-MEM and mixed gently. The wells were added to plates and incubated for 48 h until ready for further assay. All western blotting and functional studies were carried out after a 48 h-72 h transfection.
3. Construct and 6-10B cells transfection
The expression construct WT human TRPM7/pCDNA4/TO were gifts from Scharenberg Lab. This recombinant hTRPM7 is tagged with the hemagglutinin epitope.6-10B cells were transfected with the hTRPM7/pCDNA4/TO construct and empty construct by using Lipofectamine 2000 and selected for stable transfectants by zeocin treatment. TRPM7 expression was induced 1 day before use by adding 1μg/ml doxycycline to the culture medium.6-10B cells transfected with the empty vector served as a control for all experiments. Western blot analysis, wound healing assay and transwell chamber migration assay were performed 16-24 h after induction.
4. Calcium imaging
Calcium-imaging experiments were performed as described previously. Briefly, cells were plated onto glass coverslips and loaded with Fura-2 AM. Cells were then placed in a perfusion chamber on the stage of the microscope with a 40×objective. Fluorescence images of the cells were recorded by a Ratio Vision digital fluorescence microscopy system and analysed by TILL vision 4.0 software.
5. Wound healing assay
5-8F cells were cultured in 12 well tissue culture plates to confluence without vacant space. A pipette tip was used to scratch a wound midline of the culture well then cells were pretreated with mitomycin C. After 16 h of culture in RPMI 1640 supplemented with 2% serum, migration of cells was evaluated by measuring the difference in width of the wounds at 0 h and at 16 h.
6. Transwell chamber migration assay
The migration activities of 5-8F cells, siNC-transfected 5-8F cells, and siTRPM7-transfected 5-8F cells were assayed using transwell cell culture chambers. Cells were added to the transwell chamber. The motilities of NC and TRPM7 knockdown cells in the transwell chamber assay were stimulated by the serum alone or the serum in combination with BK. The serum in bottom wells acts as chemotatic factors and BK in both top and bottom wells acts as expected motility boosting factors. After 48 hours the cells that migrated through the membrane were counted. Cells were counted in 5 random fields and expressed as the average number of cells per field under a light microscope.
7. Statistical analysis
Statistical data are expressed as mean±SEM. When appropriate, Student's t-test and one-way ANOVA were applied. In all cases, p<0.05 was considered statistically significant. All experiments were repeated at least three times.
Results:
1. Expression of TRPM7 higher in 5-8F cells than in 6-10B cells The expression of TRPM7 in 5-8F and 6-10B cells was examined by western blot, RT-PCR and immunofluorescence staining. Levels of TRPM7—both mRNA and protein—were significantly higher in 5-8F cells than in 6-10B cells.
2. Silencing of TRPM7 inhibits migration of 5-8F cells Immunoblotting analyses showed that, within 48 h after transfection of 5-8F cells with TRPM7 siRNA No.1-3, the expression of TRPM7 protein was suppressed 65%-80%. Moreover, wound-healing assays showed that serum-induced migration of 5-8F cells was inhibited 50%-70%by TRPM7 siRNA. Finally, the motility of TRPM7 knockdown cells was analyzed using the transwell chamber assay. Like the down-regulated expression of TRPM7 and the decreased influx of Ca2+, motility was also reduced in cells transfected with TRPM7 siRNA. Compared with negative control siRNA cells, TRPM7 knockdown (siRNA1) cells showed a 68%reduction in the number of cells that crossed the membrane, indicating that TRPM7 channels were required for migration.
3. Overexpression of TRPM7 enhances migration of 6-10 cells Immunoblotting analyses showed that, within 48 h after transfection of 6-10B cells with hTRPM7/pCDNA4/TO, the expression of TRPM7 protein was improved significantly.Wound-healing assays and transwell chamber assays showed that BK and serum in combination or serum-induced migratory abilities increased 260%-460%in 6-10B cells transfected with WT-hTRPM7 construct when compared with control 6-10B cells.
4. Ca2+ influx mediated by TRPM7 potentially critical for migration of 5-8F cells Pharmacological approaches were used to verify that influx of extracellular Ca2+ affected the migration of 5-8F cells. In the wound-healing assay, removal of extracellular Ca2+ by EGTA significantly inhibited migration of 5-8F cells, indicating that influx of extracellular Ca2+ is critical for the migration of 5-8F cells. Next, wound-healing assays were employed to investigate the effect on cell migration of pharmacological inhibitors for various ion channels that mediated Ca2+ influx. Results showed that four inhibitors of three different Ca2+ channels had no effect on migration of 5-8F cells. However, the addition of a non-specific TRPM7 channel inhibitor 2-APB reduced the migration of 5-8F cells. Likewise, another non-specific TRPM7 channel inhibitor La3+ inhibited the migration of 5-8F cells. Conversely, the addition of BK, a peptide agonist that activates and promotes expression of TRPM7 in N1E-115 cells, promoted the migration of 5-8F cells. Transwell chamber assays also revealed that both 2-APB and La3+ inhibited the migration of 5-8F cells.
5. TRPM7 channel potentially a major regulator of intracellular Ca2+ response In pharmacological approaches to further confirm that TRPM7 mediated Ca2+ influx in 5-8F cells, Fura-2-based Ca2+ imaging was performed. Addition of BK triggered a rapid increase in cytosolic Ca2+ from internal stores in 5-8F cells. The initial increase in Ca2+ was transient, but was followed by a sustained phase of elevated Ca2+ that lasted for several minutes before Ca2+ returned to basal levels. Ratiometric analysis showed that compared with control (NC), silencing of TRPM7 (siRNA1) significantly decreased the transient phase of the response, but it decreased the sustained phase of the response much greater.
Fura-2-based Ca2+ imaging was also used to examine the mechanism by which TRPM7 inhibitors influenced influx of extracellular Ca2+ and release of intracellular Ca2+ stores in 5-8F cells. Both of TRPM7-mediated Ca2+ influx and Ca2+ store Ca2+ release could be induced consecutively two times by BK without any desensitization in the control group. However, compared with the control group, both La3+ and 2-APB completely inhibited the amplitude of the peak and plateau phases of the second BK-induced Ca2+ response. Interestingly, similar results were observed with EGTA, which chelated extracellular Ca2+. These data indicated that Ca2+ response was not induced by BK without extracellular Ca2+.
Moreover, ryanodine receptor inhibitor ryanodine and inositol-1,4,5-trisphosphate receptor inhibitor xestospongin C were used to investigate whether calcium-induced calcium release occurred via the RyR or IP3R in 5-8F cells. We compared the first response to BK in the absence of ryanodine with the corresponding second BK response after ryanodine pre-incubation. These results showed that ryanodine significantly reduced BK-induced Ca2+ peak responses while having no significant effect on BK-induced Ca2+ plateau phases. However, different results were obtained by inhibiting the IP3R with xestospongin C. The corresponding second BK response after xestospongin C pre-incubation was similar with the first BK response.
Taken together, these results in 5-8F cells confirm and characterize the mediating effects of TRPM7 channels, both on influx of extracellular Ca2+ and release of intracellular Ca2+ stores via RyRs, but not IP3Rs.
Conclusions:
This study supports the influx of Ca2+ as a requirement for the migration of human NPC 5-8F cells and identifies TRPM7 as a novel regulator not only of Ca2+ influx but also of 5-8F cell migration, thus showing TRPM7 as a potential prognostic indicator and therapeutic target for NPC patients.
Innovations of our study:
1. We were the first to confirm how TRPM7 channels function to regulate the migration of tumor cells.
2. Our preliminary study demonstrated TRPM7 was a key gene/protein related to migration of NPC.
3. Our study identified TRPM7 as a novel potential-regulator of the Ca2+ influx that allows migration of 5-8F cells. TRPM7, therefore, might have potential as a prognostic indicator and as a therapeutic target in nasopharyngeal carcinoma.
4. Stable TRPM7-transduced nasopharyngeal carcinoma cell line 6-10B was established, which will be provided a valuable tool for TRPM7 functional studies.
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[1]Punita Dhawan, Amar B. Singh, Natasha G. Deane, et al.Claudin-1 regulates cellular transformation and metastatic behavior in colon cancer [J]. The Journal of Clinical Investigation,2005,115(7):1765-1776
[2]高进,章静波.癌细胞转移模型的建立与应用[M].北京：科学出版社2003：4－78.
[3]Zamore PD, Tuschl T, Sharp PA, et al. RNAi:double stranded RNA directs the ATP dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell,2000, 101(1):25-33.
[4]Hannon GJ. RNA interference. Nature,2002,418 (6984):244-251.
[5]Fire A, Xu S, Montgomery MK, et al. Potent and special genetics interference by double-stranded RNA in Caenorhabditis elegans. Nature,1998,391(6669): 806-811.
[6]McManus MT, Sharp PA. Gene silencing in mammals by small interference RNAs. Nat Rev Genet,2002,3(10):737-747.
[7]Okegawa T, Pong RC, Li Y, et al. The role of cell adhesion molecules in cancer progression and its application of cancer therapy. Acta Biochem Pol,2004, 51(2):445-457.
[8]Furuse M, Hata M, Furuse K, et al.CLD-based tight junctions are crucial for the mammalian epidermal barrier:a lesson from CLD1-deficient mice [J]. Cell Biol,2002,156(6):1099-1111.
[9]Murray B, Tamako Konkin, Justin Routhier, et al. CLD1 is a strong prognostic indicator in stage Ⅱ colonic cancer:a tissue microarray study [J]. Modern Pathology,2005,18:511-518
[10]Miwa, Mikio Furuse, Shoichiro Tsukita, et al. Involvement of CLD1 in the beta-catenin/Tcf signaling pathway and its frequent upregulation in human colorectal cancers [J]. Oncol Res,2000,12(11-12):469-476.
[11]Weinstein RS, Mer FB, Alroy J. The structure and function of intercellular junctions in cancer. Cancer Res 1976; 23:23-89.
[12]Kerjaschki D, Krisch K, Sleyter UB, et al. The structure of tight junctions in human thyroid tumors. A freezefracture study. Am J Pathol,1979,96:207-225.
[13]Gupta AK, Melton LJ, Petersen GM, et al. Changing trends in the incidence, stage, survival, and screen-detection of colorectal cancer:a population-based study [J].Clin Gastroenterol Hepatol,2005,3(2):150-158.
[14]O'Connell JB, Maggard MA,Liu JH, et al. Are survival rates different for young and older patients with rectal cancer [J]. Dis Colon Rectum,2004,47 (12):2064-2069.
[15]Campo E.collagenase Merino MJ, Liotta L, et al. Distribution of the 72-kd type Ⅳ in nonneoplastic and neoplastic thyroid tissue. Hum Pathol,1992,23(12): 1395-1401.
[1]Yang J, Mani SA, Donaher J et al. A master regulator of morphogenesis, plays an esstial role in tumor metastasis. Cell,2004,117(7):927-939.
[2]MacDonald JF, Xiong ZG, Jackson MF.Paradox of Ca2+ signaling, cell death and stroke.Trends Neurosci,2006,29(2):75-81.
[3]L.M.Duncan, J.Deeds, J.Hunter, et al. Down-regulation of the novel genemelastatin correlates with potential for melanoma metastasis, Cancer Res. 58 (7)(1998)1515-1520.
[4]R.Wondergem, T.W. Ecay, F.Mahieu,G.Owsianik,B.Nilius,HGF/SF andmenthol increase human glioblastoma cell calcium and migration, Biochem. Biophys. Res. Commun.372 (2008) 210-215.
[5]J. Vriens, A. Janssens, J. Prenen, B. Nilius, R. Wondergem, TRPV channels and modulation by hepatocyte growth factor/scatter factor in human hepatoblastoma (HepG2) cells, Cell Calcium 36 (2004) 19-28.
[6]Kohn EC, Liotta LA.Molecular insights into cancer invasion atrategies for prevention and intervention [J].Cancer Res,1995; 55(9):1856-1862.
[7]Scott, Weinberg.A bifunction regulator of cellular proliferation. Glycoconjugate Journal,2004,19(7-9):467-477.
[8]Kohn EC, Liotta LA. Molecular insights into cancer invasionatrategies for prevention and intervention [J]. Cancer Res,1995; 55(9):1856-1862.
[9]Martin E, Hemler. Specific traspanin functions. J Cell Biol,2001, 7(155):1103-1108.
[1]B.T. Jacques-Fricke, Y.Seow, P.A. Gottlieb, F. Sachs, T.M. Gomez, Ca2+ influx through mechanosensitive channels inhibits neurite outgrowth in opposition to other influx pathways and release from intracellular stores, J. Neurosci.26 (2006) 5656-5664.
[2]M.J. Berridge, M.D. Bootman, H.L. Roderick, Calcium signalling:dynamics, homeostasis and remodelling, Nat. Rev. Mol. Cell Biol.4 (2003) 517-529.
[3]G.R. Monteith, D. McAndrew, H.M. Faddy, S.J. Roberts-Thomson, Calcium and cancer:targeting Ca2+ transport, Nat. Rev. Cancer 7 (2007) 519-530.
[4]S. Yang, J.J. Zhang, X.Y. Huang, Orail and STIM1 are critical for breast tumor cell migration and metastasis, Cancer Cell 15 (2009) 124-134.
[5]G. Amuthan, G. Biswas, H.K. Ananadatheerthavarada, C. Vijayasarathy, H.M. Shephard, N.G Avadhani, Mitochondrial stress-induced calcium signaling, phenotypic changes and invasive behavior in human lung carcinoma A549 cells, Oncogene21 (2002) 7839-7849.
[6]J.B. Huang, A.L. Kindzelskii, A. J. Clark, H.R. Petty, Identification of channels promoting calcium spikes and waves in HT1080 tumor cells:their apparent roles in cell motility and invasion, Cancer Res.64 (2004) 2482-2489.
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[10]C. Wei, X. Wang, M. Chen, K. Ouyang, L.S. Song, H. Cheng, Calcium flickers steer cell migration, Nature 457 (2009) 901-905.
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[1]Punita Dhawan, Amar B. Singh, Natasha G. Deane, et al.Claudin-1 regulates cellular transformation and metastatic behavior in colon cancer [J]. The Journal of Clinical Investigation,2005,115(7):1765-1776
[2]高进,章静波.癌细胞转移模型的建立与应用[M].北京：科学出版社2003：4－78.
[3]Zamore PD, Tuschl T, Sharp PA, et al. RNAi:double stranded RNA directs the ATP dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell,2000, 101(1):25-33.
[4]Hannon GJ. RNA interference. Nature,2002,418 (6984):244-251.
[5]Fire A, Xu S, Montgomery MK, et al. Potent and special genetics interference by double-stranded RNA in Caenorhabditis elegans. Nature,1998,391(6669): 806-811.
[6]McManus MT, Sharp PA. Gene silencing in mammals by small interference RNAs. Nat Rev Genet,2002,3(10):737-747.
[7]Okegawa T, Pong RC, Li Y, et al. The role of cell adhesion molecules in cancer progression and its application of cancer therapy. Acta Biochem Pol,2004, 51(2):445-457.
[8]Furuse M, Hata M, Furuse K, et al.CLD-based tight junctions are crucial for the mammalian epidermal barrier:a lesson from CLD1-deficient mice [J]. Cell Biol,2002,156(6):1099-1111.
[9]Murray B, Tamako Konkin, Justin Routhier, et al. CLD1 is a strong prognostic indicator in stage Ⅱ colonic cancer:a tissue microarray study [J]. Modern Pathology,2005,18:511-518
[10]Miwa, Mikio Furuse, Shoichiro Tsukita, et al. Involvement of CLD1 in the beta-catenin/Tcf signaling pathway and its frequent upregulation in human colorectal cancers [J]. Oncol Res,2000,12(11-12):469-476.
[11]Weinstein RS, Mer FB, Alroy J. The structure and function of intercellular junctions in cancer. Cancer Res 1976; 23:23-89.
[12]Kerjaschki D, Krisch K, Sleyter UB, et al. The structure of tight junctions in human thyroid tumors. A freezefracture study. Am J Pathol,1979,96:207-225.
[13]Gupta AK, Melton LJ, Petersen GM, et al. Changing trends in the incidence, stage, survival, and screen-detection of colorectal cancer:a population-based study [J].Clin Gastroenterol Hepatol,2005,3(2):150-158.
[14]O'Connell JB, Maggard MA,Liu JH, et al. Are survival rates different for young and older patients with rectal cancer [J]. Dis Colon Rectum,2004,47 (12):2064-2069.
[15]Campo E.collagenase Merino MJ, Liotta L, et al. Distribution of the 72-kd type Ⅳ in nonneoplastic and neoplastic thyroid tissue. Hum Pathol,1992,23(12): 1395-1401.
[1]Yang J, Mani SA, Donaher J et al. A master regulator of morphogenesis, plays an esstial role in tumor metastasis. Cell,2004,117(7):927-939.
[2]MacDonald JF, Xiong ZG, Jackson MF.Paradox of Ca2+ signaling, cell death and stroke.Trends Neurosci,2006,29(2):75-81.
[3]L.M.Duncan, J.Deeds, J.Hunter, et al. Down-regulation of the novel genemelastatin correlates with potential for melanoma metastasis, Cancer Res. 58 (7)(1998)1515-1520.
[4]R.Wondergem, T.W. Ecay, F.Mahieu,G.Owsianik,B.Nilius,HGF/SF andmenthol increase human glioblastoma cell calcium and migration, Biochem. Biophys. Res. Commun.372 (2008) 210-215.
[5]J. Vriens, A. Janssens, J. Prenen, B. Nilius, R. Wondergem, TRPV channels and modulation by hepatocyte growth factor/scatter factor in human hepatoblastoma (HepG2) cells, Cell Calcium 36 (2004) 19-28.
[6]Kohn EC, Liotta LA.Molecular insights into cancer invasion atrategies for prevention and intervention [J].Cancer Res,1995; 55(9):1856-1862.
[7]Scott, Weinberg.A bifunction regulator of cellular proliferation. Glycoconjugate Journal,2004,19(7-9):467-477.
[8]Kohn EC, Liotta LA. Molecular insights into cancer invasionatrategies for prevention and intervention [J]. Cancer Res,1995; 55(9):1856-1862.
[9]Martin E, Hemler. Specific traspanin functions. J Cell Biol,2001, 7(155):1103-1108.
[1]B.T. Jacques-Fricke, Y.Seow, P.A. Gottlieb, F. Sachs, T.M. Gomez, Ca2+ influx through mechanosensitive channels inhibits neurite outgrowth in opposition to other influx pathways and release from intracellular stores, J. Neurosci.26 (2006) 5656-5664.
[2]M.J. Berridge, M.D. Bootman, H.L. Roderick, Calcium signalling:dynamics, homeostasis and remodelling, Nat. Rev. Mol. Cell Biol.4 (2003) 517-529.
[3]G.R. Monteith, D. McAndrew, H.M. Faddy, S.J. Roberts-Thomson, Calcium and cancer:targeting Ca2+ transport, Nat. Rev. Cancer 7 (2007) 519-530.
[4]S. Yang, J.J. Zhang, X.Y. Huang, Orail and STIM1 are critical for breast tumor cell migration and metastasis, Cancer Cell 15 (2009) 124-134.
[5]G. Amuthan, G. Biswas, H.K. Ananadatheerthavarada, C. Vijayasarathy, H.M. Shephard, N.G Avadhani, Mitochondrial stress-induced calcium signaling, phenotypic changes and invasive behavior in human lung carcinoma A549 cells, Oncogene21 (2002) 7839-7849.
[6]J.B. Huang, A.L. Kindzelskii, A. J. Clark, H.R. Petty, Identification of channels promoting calcium spikes and waves in HT1080 tumor cells:their apparent roles in cell motility and invasion, Cancer Res.64 (2004) 2482-2489.
[7]E.J. Pettit, F.S. Fay, Cytosolic free calcium and the cytoskeleton in the control of leukocyte chemotaxis, Physiol. Rev.78 (1998) 949-967.
[8]J. Lee, A. Ishihara, G. Oxford, B. Johnson, K. Jacobson, Regulation of cell movement is mediated by stretch-activated calcium channels, Nature 400 (1999) 382-386.
[9]G. Giannone, P. Ronde, M. Gaire, J. Haiech, K. Takeda, Calcium oscillations trigger focal adhesion disassembly in human U87 astrocytoma cells, J. Biol. Chem.277 (2002) 26364-26371.
[10]C. Wei, X. Wang, M. Chen, K. Ouyang, L.S. Song, H. Cheng, Calcium flickers steer cell migration, Nature 457 (2009) 901-905.
[11]K. Clark, M. Langeslag, B. van Leeuwen, et al., TRPM7, a novel regulator of actomyosin contractility and cell adhesion, EMBO J.25 (2006) 290-301.
[12]L.M. Duncan, J. Deeds, J. Hunter, et al., Down-regulation of the novel gene melastatin correlates with potential for melanoma metastasis, Cancer Res.58 (7) (1998) 1515-20.
[13]L.M. Duncan, J. Deeds, F.E. Cronin, et al., Melastatin expression and prognosis in cutaneous malignant melanoma, J. Clin. Oncol.19 (2001) 568-576.
[14]L. Tsavaler, M.H. Shapero, S. Morkowski, R. Laus, Trp-p8, a novel prostate-specific gene, is up-regulated in prostate cancer and other malignancies and shares high homology with transient receptor potential calcium channel proteins, Cancer Res.61 (2001) 3760-3769.
[15]G. Bidaux, M. Flourakis, S. Thebault, et al., Prostate cell differentiation status determines transient receptor potential melastatin member 8 channel subcellular localization and function, J. Clin. Invest.117 (2007) 1647-1657.