瞬时受体电位通道TRPM8在人乳腺癌细胞中的表达及其对细胞迁移能力的影响
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摘要
恶性肿瘤是一种临床上常见、高发、难早诊断、难治愈、严重危害人类生命和健康的慢性疾病,现已成为全球重大的公共卫生问题。研究表明中国2009年全国肿瘤登记地区恶性肿瘤发病率为285.91/10万,恶性肿瘤死亡率为180.54/10万。2009年中国女性恶性肿瘤发病第1位的是乳腺癌,转移性乳腺癌是女性常见的恶性肿瘤之一,全球每年约有120万妇女发生乳腺癌,其中有一半左右的患者在10年内死亡,国内转移性乳腺癌的发病率呈现递增的趋势,已成为部分城市妇女中占首位的恶性肿瘤,而且发病年龄越来越趋向于年轻化。恶性肿瘤给我国人民的生命和健康造成巨大威胁,给社会和家庭带来巨大经济压力和心里负担。
恶性肿瘤的治疗方法主要是手术治疗、放射治疗、化学治疗、生物治疗、介入治疗、射频治疗、冷冻治疗、激光治疗及中医药治疗等,但肿瘤细胞的耐药性及转移仍然是严重遏制恶性肿瘤治疗有效性的主要因素。侵袭和转移是恶性肿瘤患者的重要死因,90%以上的恶性肿瘤患者最终死于肿瘤转移或者复发,现有的常规治疗手段尚无法从根本上清除恶性肿瘤,了解恶性肿瘤侵袭、转移发生的关键机制,揭示恶性肿瘤侵袭及转移的分子作用机制及可能的干预措施也成为当今国、内外肿瘤基础及临床研究热点。
肿瘤侵袭与转移过程非常复杂,尽管不同种类肿瘤的侵袭、转移特性有所不同,但侵袭、转移过程中的关键步骤基本相同。侵袭是指恶性肿瘤细胞侵犯和破坏周围正常组织进入循环系统的过程。转移是指侵袭中的癌细胞迁移到特定组织器官并发展成为继发性癌灶的过程。侵袭和转移是同一过程中的两个不同阶段,侵袭是转移的前奏,转移是侵袭的结果。
肿瘤转移包括粘附、侵袭和迁移三个过程,对于肿瘤转移的机理,Liotta提出的癌细胞侵袭、转移的三步骤假说是目前较为认可的。三步骤假说：①肿瘤细胞之间的解黏附和肿瘤细胞与细胞外基质(extracellular matrix, 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。
深刻理解肿瘤细胞侵袭和转移的分子机制将为诊断和治疗恶性肿瘤提供新的策略,抑制肿瘤的侵袭、转移过程将会成为肿瘤治疗的新靶点。
近10年来,瞬时受体电位通道(transient receptor potential channels,TRP channels)与恶性肿瘤的关系在某种程度上已越来越明确,TRP通道参与受体激活、细胞异常增殖、分化、损伤乃至死亡,从而导致恶性肿瘤细胞不可控制的增殖和侵袭。越来越多的学者致力于TRP通道的生理功能及其在癌症发生、发展中的变化方面的研究。
瞬时受体电位通道M型(melastatin-related transient receptor potential,TRPM)是位于细胞膜上的一类重要的阳离子通道家族。TRPM8分布广泛,TRPM8在某些肿瘤的发生、发展中起重要作用。已有研究表明TRPM7与乳腺癌的转移、侵袭有关,也有研究结果表明TRPM8在人乳腺癌组织中过度表达,而TRPM8也已经成为前列腺癌病人预后不良的一个临床指标,TRPM8的作用机制研究为肿瘤基础研究提供思路和线索。
本研究试图从TRPM8在乳腺癌组织、乳腺正常组织的表达差异及运用基因沉默和稳定转染技术初步研究TRPM8在乳腺癌细胞迁移中的可能作用来探讨TRPM8作为抑制乳腺癌转移的分子靶点的可能性,为乳腺癌早期诊断、治疗评价及判断预后提供参考。
方法
1.检测人体乳腺癌旁组织与乳腺癌组织中TRPM8基因、蛋白水平表达
选取4组配对的乳腺癌旁组织及癌组织标本为研究受试对象。采用QPCR、 Western Blot方法分别在mRNA.蛋白表达水平检测TRPM8在人体乳腺癌旁组织与乳腺癌组织之间的表达量是否存在差异。
2.检测人正常乳腺上皮细胞株与人乳腺癌细胞株TRPM8基因、蛋白水平表达选取正常乳腺上皮细胞株MCF-10A以及不同转移能力乳腺癌细胞株MCF-7、T47D. MDA-MB-231、BT549、SKBR3、ZR-75-30为研究受试对象。采用QPCR、 Western Blot方法分别在mRNA、蛋白表达水平检测TRPM8在正常乳腺上皮细胞株MCF-10A及不同转移能力乳腺癌细胞株中的表达差异。
3.RNAi干扰TRPM8基因对人乳腺癌高转移细胞株MDA-MB-231迁移能力的影响利用基因公司设计合成的3条靶向TRPM8基因的siRNA片段(用TRPM8-RNAi-1、RPM8-RNAi-2和TRPM8-RNAi-3表示)和1条阴性对照RNAi片段(用Scramble表示),分别将4条siRNA片段转染进入不同的MDA-MB-231细胞,利用QPCR和Western Blot方法分别检测各组细胞的]PRPM8表达水平,筛选出TRPM8-RNAi-1-3中干扰效率最高的siRNA片段。接着利用RNA干扰(RNAi)技术干扰高表达TRPM8的高转移能力的MDA-MB-231细胞株其TRPM8mRNA表达量,采用QPCR. Western Blot方法检测TRPM8基因干扰效果,采用三维培养模型观察干扰细胞的形态改变,分别用细胞划痕实验及transwell小室实验检测已成功干扰TRPM8的人乳腺癌细胞MDA-MB-231/siTRPM8的迁移能力变化。
4.稳定转染TRPM8基因对人乳腺癌低转移细胞株MCF-7迁移能力的影响
将TRPM8目的基因序列克隆入pBABE-puro表达载体,建立pBABE-puro-TRPM8表达载体。经限制性内切酶酶切鉴定、测序并转染293FT细胞判断成功构建pBABE-puro-TRPM8表达载体。将空质粒和pBABE-puro-TRPM8表达载体分别转染进入人乳腺癌MCF-7细胞,建立稳定表达TRPM8的MCF-7/pBABE-puro-TRPM8细胞,应用QPCR、Western Blot检测稳定转染细胞的TRPM8基因及蛋白表达水平；采用三维培养模型观察稳定转染TRPM8基因的细胞形态改变,并运用细胞划痕实验、transwell小室实验检测稳定转染TRPM8基因的MCF-7细胞的迁移能力变化。
5.TRPM8影响MCF-7、MDA-MB-231细胞迁移能力机制的初步探讨
选取沉默内源性TRPM8基因的MDA-MB-231细胞及稳定转染TRPM8基因的MCF-7细胞为受试细胞,采用QPCR、Western blot分别在mRNA、蛋白表达水平检测沉默TRPM8前、后MDA-MB-231细胞及转染TRPM8前、后MCF-7细胞中E-cadherin、Vimentin、Fibronectin、AKT、p-AKT、GSK-3β、p-GSK-3β、 Snail的表达变化,以探讨TRPM8基因对人乳腺癌细胞迁移能力的影响及其相关机制。
6.统计学方法：
采用SPSS21.0统计软件进行处理,计量资料用均数±标准差(x±s)表示,若方差齐性采用ONE-WAY ANOVA,多重比较采用LSD法,若方差不齐,整体比较采用Welch检验,多重比较则采用Dunnett's T3检验,以P<0.05为差异有统计学意义；mRNA检测则采用单样本t检验,以P<0.05为差异有统计学意义；两组比较,采用两独立样本t检验,以P<0.05为差异有统计学意义。
结果
1.TRPM8在人体乳腺癌旁组织和乳腺癌组织mRNA、蛋白水平的表达：
4例标本的检测结果均表明人乳腺癌组织中TRPM8在mRNA、蛋白表达水平上均较其在人乳腺癌旁组织中的表达水平高,人体乳腺癌组织和人乳腺癌旁组织其TRPM8mRNA及蛋白表达差异均有统计学意义(P<0.05)。
2.TRPM8在人正常乳腺上皮细胞株与人乳腺癌细胞株mRNA、蛋白水平的表达：
QPCR和Western blot检测正常乳腺上皮细胞株MCF-10A及6种人乳腺癌MCF-7、T47D、MDA-MB-231、BT549、SKBR3、ZR-75-30细胞中TRPM8在mRNA和蛋白水平的相对表达量,两种检测方法的结果基本一致：①在mRNA及蛋白水平,6种人乳腺癌细胞的TRPM8相对表达水平都高于其在正常乳腺上皮细胞的表达水平；②正常乳腺上皮细胞株MCF-10A的TRPM8相对表达水平最低；③高转移能力乳腺癌细胞MDA-MB-231的TRPM8表达水平最高；④低转移能力乳腺癌细胞MCF-7的TRPM8表达水平相对较低；⑤各乳腺癌细胞与正常乳腺上皮细胞株MCF-10A之间分别在TRPM8mRNA、蛋白水平的表达量比较差异有统计学意义(P<0.05)；⑥6株乳腺癌细胞株间的TRPM8mRNA、蛋白相对表达水平多重比较,差异均有统计学意义(P<0.05)。
3.沉默TRPM8基因对人乳腺癌细胞株MDA-MB-231迁移能力的影响：
干扰TRPM8基因效率最高的干扰片段是siRNA3(约80%)；稳转TRPM8RNAi-3的MDA-MB-231细胞其TRPM8mRNA、蛋白表达水平下降,细胞间连接比较紧密,克隆边缘较光滑,未见明显的伪足。
划痕实验结果表明在Oh时MDA-MB-231/Scramble组和MDA-MB-231/siTRPM8组细胞的划痕面积基本相等,在24h时MDA-MB-231/Scramble组的细胞划痕面积小于MDA-MB-231/siTRPM8组细胞划痕面积,在48h时MDA-MB-231/Scramble组的细胞划痕面积小于MDA-MB-231/siTRPM8组细胞划痕面积。随观察时间的推移,两组细胞其无细胞划痕区逐渐变窄,沉默了TRPM8的MDA-MB-231细胞其细胞迁移能力减弱。
Transwell小室实验结果显示MDA-MB-231/siTRPM8的穿孔细胞数少于MDA-MB-231/Scramble的穿孔细胞数,与MDA-MB-231/Scramble细胞比较,MDA-MB-231/siTRPM8细胞的迁移能力降低(t=5.043,P=0.006),实验初步表明沉默TRPM8基因可能抑制了MDA-MB-231细胞的迁移能力。
通过划痕实验和Transwell小室实验得出干扰TRPM8表达后的MDA-MB-231细胞侵袭、迁移能力被抑制,这说明TRPM8可能促进乳腺癌细胞的转移。
4.TRPM8基因过表达对人乳腺癌细胞株MCF-7迁移能力的影响：
稳定转染pBABE-puro-TRPM8表达载体的原来低侵袭能力的MCF-7细胞经QPCR, Western Blot方法检测其TRPM8的表达水平,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,将其命名为MCF-7/TRPM8细胞,而对照组细胞命名为MCF-7/vector细胞。实验结果表明MCF-7/vector组与MCF-7/TRPM8组细胞其TRPM8mRNA、蛋白表达水平差异有统计学意义(P<0.05),结果提示TRPM8表达水平在MCF-7/TRPM8细胞呈上调,初步证实MCF-7/TRPM8细胞已成功转染TRPM8基因。
在细胞三维培养模型中,MCF-7/TRPM8细胞可见克隆伸出伪足向四周浸润,边界不整齐,细胞间连接较松散,从其细胞形态学改变提示乳腺癌稳定转染TRPM8基因的细胞株MCF-7/TRPM8的侵袭能力增强。
划痕实验结果表明在24h时、48小时MCF-7/TRPM8组的细胞划痕面积均小于MCF-7/vector组细胞划痕面积,随观察时间的推移,两组细胞无细胞划痕区逐渐变窄,稳定转染TRPM8的MCF-7细胞其细胞迁移能力增强。
Transwell小室实验结果显示MCF/TRPM8的穿孔细胞数高于MCF-7/Vector的穿孔细胞数,其差异有统计学意义(t=-13.058,P<0.001),实验初步表明稳定转染TRPM8基因后可能增强了MCF-7细胞的迁移能力。
5.TRPM8的表达高、低对人乳腺癌细胞株MCF-7、MDA-MB-231迁移影响的作用机制：
本实验结果表明在RNA和蛋白水平上,均显示稳定转染TRPM8的MCF-7细胞,其上皮标志物E-cadherin的表达量下调,而Vimentin、Fibronectin、Snail的表达量上调；免疫荧光实验提示乳腺癌MCF-7/Vector细胞的细胞膜上高表达E-cadherin蛋白,而其胞浆内Vimentin蛋白呈低表达；而乳腺癌MCF-7/TRPM8细胞的细胞膜上E-cadherin蛋白呈低表达,其细胞胞浆内、Vimentin蛋白则呈高表达。
在mRNA和蛋白水平上,MDA-MB-231/siTRPM8细胞的上皮标志物E-cadherin的表达量均增强,而其Vimentin、Fibronectin、Snail表达均不同程度减弱；免疫荧光实验提示乳腺癌MDA-MB-231/siTRPM8细胞的细胞膜上E-cadherin蛋白呈高表达,其胞浆内Vimentin蛋白则呈低表达；而乳腺癌MDA-MB-231/Scramble细胞的细胞膜上E-cadherin蛋白呈低表达,而其胞浆内Vimentin蛋白呈高表达。
在MDA-MB-231/siTRPM8细胞,其Akt、GSK-3β的mRNA、蛋白表达均无改变,但其Akt的磷酸化水平下降,随着p-AKT蛋白表达量的减少,GSK-3β的磷酸化水平亦呈下降趋势,P-GSK-3β蛋白表达量减少；而在稳定转染TRPM8基因的MCF-7细胞中AKt及GSK-3βmRNA、蛋白亦无改变,但其Akt的磷酸化水平(p-AKT)蛋白表达量增加,下游靶蛋白GSK-3β的磷酸化水平亦同步呈上升趋势,即P-GSK-3β蛋白表达量升高。
结论
1.人乳腺癌组织中存在TRPM8过表达的现象；
2.乳腺癌细胞中TRPM8的mRNA及蛋白相对表达量的高低与其细胞侵袭、转移能力可能有关,即细胞中的TRPM8相对表达量越高,其侵袭、转移能力越强；
3.沉默TRPM8基因的MDA-MB-231细胞(MDA-MB-231/siTRPM8)侵袭、迁移能力被抑制,这说明TRPM8可能促进乳腺癌细胞的转移；
4.细胞TRPM8表达水平的高低可能是细胞迁移能力强弱的标志,TRPM8基因过表达可能增强MCF-7细胞的迁移能力,乳腺癌细胞中TRPM8表达量越高,其细胞迁移能力越强,TRPM8可能是乳腺癌的一个转移相关基因；
5.稳定转染TRPM8基因,可能会增强低侵袭性乳腺癌MCF-7细胞发生EMT现象而增强肿瘤细胞的侵袭、转移能力,而沉默TRPM8基因可能会抑制高侵袭性乳腺癌MDA-MB-231细胞发生EMT现象而抑制肿瘤细胞的侵袭、转移能力,TRPM8增强肿瘤细胞的侵袭、转移能力的作用机制可能与其通过Akt/GSK-3p信号转导通路参与EMT过程有关。
本研究的创新之处
1.验证了乳腺癌组织中存在TRPM8过表达现象；
2.利用重组质粒pSuper-retro-puro-siTRPM8转染MDA-MB-231细胞株,筛选出稳定沉默TRPM8基因的MDA-MB-231/siTRPM8细胞株；成功构建pBABE-puro-TRPM8表达载体,转染人乳腺癌MCF-7细胞,建立了TRPM8基因过表达的稳定人乳腺癌细胞株MCF-7/TRPM8,为研究TRPM8基因的功能提供了有价值的研究工具。
3.发现TRPM8可能与乳腺癌细胞迁移相关,为开展乳腺癌基因靶向治疗初步奠定了理论基础。
4.首次阐明了TRPM8可能通过Akt/GSK-3β信号通路影响乳腺癌细胞EMT从而影响肿瘤细胞的迁移。
Malignant tumor is common chronic disease, which has become a significant public health problems worldwide. Research shows that malignant tumor incidence rate of285.91/10million at2009in China, the malignant tumor mortality rate was180.54/10million. Breast cancer is the most common form of cancer in women in western countries, and already taking the first place of incidence in chinese female cancer patients at2009. Metastatic breast cancer is one of the most common malignant tumors, there are about one million two hundred thousand women with breast cancer around the world every year, half of the patients died within10years.
Methods in the treatment of malignant tumor include surgical operation, radiotherapy, chemotherapy, biologic therapy, interventional therapy, radiofrequency therapy, cryotherapy, laser therapy and Chinese medicine treatment, but the drug resistance and metastasis of tumor cells are still the main factors that hinder the effectiveness of treatment of malignant tumor. Invasion and metastasis is the important cause of death in patients with malignant tumor, more than90%of the patients died of the tumor metastasis or recurrence, routine treatments cannot fundamentally eliminate malignant tumor. Nowadays the basic and clinical researches of tumor focus on the mechanism of malignant tumor invasion and metastasis.
Tumor invasion and metastasis process is very complex, even though different kinds of tumors invasion and metastasis have their own different characteristics. The key steps are basically the same in the process of invasion and metastasis of tumor.
In the last10years, the extent to which TRP channels are associated with cancer has been increasingly clarified, and the involvement of these receptors in triggering enhanced proliferation, aberrant differentiation, and impaired ability to die, thus leading to the uncontrolled cancer expansion and tumor invasion have been recognized. More and more scholars work on the TRP channel in the changes of physiological function and cancer.
TRPM channel is a non-selective cation channel superfamily, TRPM8belongs to the TRPM (melastatin) subfamily of TRP proteins. TRPM8is widely distributed, it plays an important role in the occurrence and development of some tumors. Studies had shown that TRPM7was related with the invasion and metastasis of breast cancer, also some researches had indicated that over expression of TRPM8was observed in human breast cancer tissues, and TRPM8has become a clinical index of the bad prognosis of prostate cancer patients, study on the mechanism of TRPM8will provide clues for the study of tumor.
This study attempted to explore the possibility of TRPM8as a molecular target in inhibiting metastasis of breast cancer, TRPM8channel might represent valuable diagnostic and/or prognostic markers, as well as targets for pharmaceutical intervention and targeting in cancer.
Methods
1. Tissue samples
Primary cancer tissues and adjacent non-tumor tissues were obtained from surgical treatments of breast cancer. All samples were formalin-fixed and paraffin-embedded (FFPE) with standard procedures. The histological diagnosis was made by a pathologist and has been re-confirmed by a second pathologist. The study was approved by the Institutional Review Board (IRB) in the Southern Medical University and consented by patients involved.
2. Cell culture
Breast epithelial cell line MCF-10A and metastatic breast cancer cell lines including MCF-7, T47D, MDA-MB-231, BT549, SKBR3and ZR-75-30were purchased from American Type Culture Collection (ATCC) and preserved in Central Laboratory in Southern Medical University. Each cell line is maintained in culture medium and atmosphere instructed by each manuscripts.
3. Generation of stable cell lines
The sequence of TRPM8and shRNA-TRPM8were cloned into pMSCV-puromycin and pSuper-puromycin retroviral vectors respectively, and the resulting plasmid was transfected into293FT cells to generate virus as described previously. After incubation at37℃for6h, the transfected cells were cultured in fresh media overnight. In the following days, media were collected three times a day to gather the produced virus until the293FT cells reached total confluency. Media containing pMSCV/TRPM8or pSuper/shRNA-TRPM8were used to infect MCF-7or MDA-MB-231respectively for24h. After removal of the inoculum and replacement with fresh media, infected cells were selected by adding puromycin. Stable cell lines were verified by Western blotting and qRT-PCR.
4. Quantitative Real Time-PCR (qRT-PCR) analysis
Total RNA from different cell lines and human tissues was extracted using Trizol reagent. cDNA was synthesized from2.5μg of total RNA using random hexamers. Real-time PCR was carried out using an ABI PRISM7500Sequence Detection System. Reactions were run in triplicate in three independent experiments.The geometric mean of the housekeeping gene β-actin was used as internal control to normalize the variability in expression levels.
5. Wound healing assay
One day before scratch, MCF-7/TRPM8and MDA-MB-231/RNAi cells were trypsinized and seeded equally into6-well tissue culture plates, and grew to reach almost total confluence in24h. When non-serum starvation kept for24h after cell monolayer formed, an artificial homogenous wound was created onto the monolayer with a sterile200μL tip. After scratching, the cells were washed with serum-free medium. Images of cells migrating into the wound were captured at time points of0h,24h and48h by inverted microscope (200×).
6. In vitro invasion assay
The invasion assay was done by using Transwell chamber consisting of8mm membrane filter inserts coated with Matrigel as previously described. Briefly, cells were trypsinized and suspended in serum-free medium. Then1×104cells were added to the upper chamber, whereas lower chamber was filled with medium with10%FBS. After incubated for48h, cells were invaded through the coated membrane to the lower surface, in which cells were fixed with4%paraformaldehyde and stained with hematoxylin. The cell count was done under the microscope (200×).
7. Western blotting
For the expression analysis of EMT-related proteins, immunoblotting assay was carried out. MCF-7/TRPM8and MDA-MB-231/RNAi cells were seeded in100mm tissue culture dishes. After24h, cells were washed with prechilled PBS when the confluence reached to60-70%, followed by being harvested in sample buffer [62.5mmol/L Tris-HCl (pH6.8),2%SDS,10%glycerol, and5%2-b-mercaptoethanol]. Equal amounts of protein from the supernatant were loaded per lane and resolved by SDS-polyacrylamide electrophoresis. In sequence, protein was transferred onto PVDF membrane, blocked by5%nonfat milk for1h at room temperature, and probed with primary antibodies for3h. Primary antibodies for E-Cadherin, Fibronectin, Vimentin and Snail were purchased from BD Biosciences. Those for AKT, p-AKT, GSK-3β p-GSK-3β were purchased from Cell Signaling Technology. Membranes were washed thrice (10min each) in TBS-T buffer and incubated for40min at room temperature with horseradish peroxidase-conjugated anti-mouse secondary antibodies. Blots were washed thrice (10min each) in TBS-T and developed using the ECL system. Protein loading was normalized by reprobing the blots with a-Tubulin antibody.
8. Immunofluorescence analysis
Cells were stained for immunofluorescence on coverslips as described previously. Briefly, the cells were incubated with primary antibodies against E-cadherin and Vimentin, and then incubated with rhodamine-conjugated or FITC-conjugated goat antibodies against rabbit or mouse IgG. The coverslips were counterstained with DAPI and imaged with a confocal laser-scanning microscope.
9.3D morphogenesis assay
Twenty-four-well dishes were coated with Growth Factor Reduced Matrigel and covered with growth medium supplemented with2%Matrigel as previously described. Cells were trypsinized and seeded at a density of104cells/well, and medium was replaced with2%Matrigel for3to4days. Microscopic images were captured at2-day intervals for2-3weeks.
10. Statistical analysis
Statistics were assessed using SPSS21.0(SPSS, Inc., Chicago, IL, USA). Statistical data are expressed as mean±SEM, When appropriate, Student's t-test and one-way ANOVA were applied. In all cases, P<0.05was considered statistically significant. All experiments were repeated at least three times.
Results
1. The expressions of TRPM8in human breast adjacent non-tumor tissues and tumor tissues.
Four pairs of resected breast tissues were included to our study. TRPM8protein levels were obviously higher in tumor tissues than in the pairing adjacent non-tumor tissues. To consolidate this finding, we detected mRNA expressions of TRPM8in each sample pairs, and result is consistent with of protein level.
2. The expressions of TRPM8in one human breast normal epithelial cell and in six human breast cancer cells.
Expression of TRPM8was examined at transcription and protein level in one breast normal epithelial cell line and in six cancer cell lines using western blotting and qRT-PCR. A very strong to moderate expression of TRPM8was observed in all cell lines except for MCF-10A cell that showed very low expression which was normalized as base line in qRT-PCR. Among the six breast cancer cell lines, MDA-MB-231showed the highest expression of TRPM8, while MCF-7, T47D and ZR-75-30demonstrated relatively lower TRPM8levels
3. Effects of TRPM8gene silencing on the migration of human breast cancer cell line MDA-MB-231
We interfered endogenous expression of TRPM8in MDA-MB-231by transfecting silencing plasmids, expression of TRPM8decreased at transcription and protein level in MDA-MB-231/siTRPM8. Morphologically, low expression of TRPM8induced major3D-shape changes in comparison to control cells, The cells connections of MDA-MB-231/siTRPM8were closer, the edge of MDA-MB-231/siTRPM8was smoother, there was not obvious pseudopodia in MDA-MB-231/siTRPM8.
Cell migration observed by wound healing assay, cells transfected with siTRPM8moved more slowly than those with scramble sequence.The results of Transwell chamber experiments showed that MDA-MB-231/siTRPM8cells penetrated through the gel-membrane section significantly less than MDA-MB-231/Scramble, The migration ability of MDA-MB-231/siTRPM8cells decreased significantly(t=5.043, P=0.006), The preliminary results showed that TRPM8gene silencing could inhibit the migration of MDA-MB-231cells.
4. Effects of TRPM8gene over-expression on the migration of human breast cancer MCF-7cell
To investigate the role of TRPM8in the development and progression of breast cancer, TRPM8over-expression cells were established by plasmid transfection to perform migration and invasion assays in vitro. The experimental results showed that TRPM8expression level was upregulated in MCF-7/TRPM8cells, which confirmed that MCF-7/TRPM8cells have been successfully transfected with TRPM8gene.
As we expect, cell migration observed by wound healing assay demonstrated that cells transfected with TRPM8plasmid significantly ran faster than those with vector control.
Three dimensional cell culture model showed MCF-7/TRPM8with the formation of thorns or pseudopodia invaded the surrounding, which suggested MCF-7/TRPM8had more motile and higher invasiveness. Cell migration observed by wound healing assay, MCF-7cells transfected with TRPM8moved more faster than those with empty vector.
The invasive property of MCF-7cells was examined by Transwell-Matrigel penetration assay, which depicted cells transfected with TRPM8penetrated through the gel-membrane section much more than cells transfected with vector(MCF-7/Vector). The preliminary results showed that stable transfection of TRPM8gene could enhance the migration ability of MCF-7cells.
5. The mechanism of TRPM8how to effect on the migration of MCF-7and MDA-MB-231
To investigate whether TRPM8regulates malignancy and metastatic features of breast cancer cells by promoting EMT, we interfered endogenous expression of TRPM8in MDA-MB-231by transfecting silencing plasmids while increased TRPM8level in MCF-7by introducing TRPM8-expressing constructs, over-expression of TRPM8repressed E-cadherin, one of epithelial markers and supposed to be down-regulated during EMT. Nevertheless, E-cadherin was increased in breast cancer cells transfected with TRPM8silencing plasmids. Moreover, Fibronectin and Vimentin, which are mesenchymal markers and should be up-regulated during EMT, were repressed in TRPM8-silenced cells. However, they were significantly increased in cells transfected with TRPM8plasmid. In order to observe visually, we probed the cells by immunofluorescent staining. cells with high TRPM8exhibited obvious down-regulation of E-cadherin and up-regulation of Vimentin, vice versa. These suggested that TRPM8had positive effects on EMT in breast cancer and might conduct epithelial cells to transform toward mesenchymal cells.
Changing TRPM8expression did not obviously influence the expression levels of Akt and GSK-3β, but phosphorylation of Akt was significantly inhibited in cells transduced with siRNA against TRPM8(MDA-MB-231/siTRPM8). The increase in Akt phosphorylation was accompanied by a change in phosphorylation of GSK-3P, a downstream target protein of Akt, suggesting that up-regulation of TRPM8activates the Akt/GSK-3β pathway in breast cancer cells. Western blotting revealed that, compared with vector cells, expression of Snail significantly increased in breast cancer cells transfected with TRPM8plasmid but decreased in those with silencing constructs.
Conclusions
1. TRPM8was highly expressed in human breast cancer tissues.
2. TRPM8was a key gene/protein related to migration of human breast cancer, increasing the expression of TRPM8would enhance the migration of breast cancer cells.
3. TRPM8gene silencing inhibited the invasion and migration of MDA-MB-231, which indicated TRPM8might promote the migration of breast cancer cell.
4. TRPM8may be a metastatic related gene in breast cancer.
5.TRPM8enhances the invasion and migration of tumor cells, whose mechanism may be involved in EMT process by the Akt/GSK-3β signaling pathway.
Innovations of our study
1. This study has verified that over-expression of TRPM8exists in breast cancer tissues.
2. Stable silencing TRPM8cell line MDA-MB-231/siTRPM8was established with pSuper-retro-puro-siTRPM8transfection and puro selection, and stable TRPM8-transduced breast cancer line MCF-7was established with pBABE-puro-TRPM8, which would be provided a valuable tool for TRPM8functional studies.
3. TRPM8could be related to the migration of human breast cancer, which would provide theoretical basis on targeted therapy of breast cancer.
4. The preliminary study demonstrated how TRPM8channels function to regulate the migration of breast cancer cells by Akt/GSK-3β pathway which affected human breast cancer EMT.
恶性肿瘤的治疗方法主要是手术治疗、放射治疗、化学治疗、生物治疗、介入治疗、射频治疗、冷冻治疗、激光治疗及中医药治疗等,但肿瘤细胞的耐药性及转移仍然是严重遏制恶性肿瘤治疗有效性的主要因素。侵袭和转移是恶性肿瘤患者的重要死因,90%以上的恶性肿瘤患者最终死于肿瘤转移或者复发,现有的常规治疗手段尚无法从根本上清除恶性肿瘤,了解恶性肿瘤侵袭、转移发生的关键机制,揭示恶性肿瘤侵袭及转移的分子作用机制及可能的干预措施也成为当今国、内外肿瘤基础及临床研究热点。
肿瘤侵袭与转移过程非常复杂,尽管不同种类肿瘤的侵袭、转移特性有所不同,但侵袭、转移过程中的关键步骤基本相同。侵袭是指恶性肿瘤细胞侵犯和破坏周围正常组织进入循环系统的过程。转移是指侵袭中的癌细胞迁移到特定组织器官并发展成为继发性癌灶的过程。侵袭和转移是同一过程中的两个不同阶段,侵袭是转移的前奏,转移是侵袭的结果。
肿瘤转移包括粘附、侵袭和迁移三个过程,对于肿瘤转移的机理,Liotta提出的癌细胞侵袭、转移的三步骤假说是目前较为认可的。三步骤假说：①肿瘤细胞之间的解黏附和肿瘤细胞与细胞外基质(extracellular matrix, 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。
深刻理解肿瘤细胞侵袭和转移的分子机制将为诊断和治疗恶性肿瘤提供新的策略,抑制肿瘤的侵袭、转移过程将会成为肿瘤治疗的新靶点。
近10年来,瞬时受体电位通道(transient receptor potential channels,TRP channels)与恶性肿瘤的关系在某种程度上已越来越明确,TRP通道参与受体激活、细胞异常增殖、分化、损伤乃至死亡,从而导致恶性肿瘤细胞不可控制的增殖和侵袭。越来越多的学者致力于TRP通道的生理功能及其在癌症发生、发展中的变化方面的研究。
瞬时受体电位通道M型(melastatin-related transient receptor potential,TRPM)是位于细胞膜上的一类重要的阳离子通道家族。TRPM8分布广泛,TRPM8在某些肿瘤的发生、发展中起重要作用。已有研究表明TRPM7与乳腺癌的转移、侵袭有关,也有研究结果表明TRPM8在人乳腺癌组织中过度表达,而TRPM8也已经成为前列腺癌病人预后不良的一个临床指标,TRPM8的作用机制研究为肿瘤基础研究提供思路和线索。
本研究试图从TRPM8在乳腺癌组织、乳腺正常组织的表达差异及运用基因沉默和稳定转染技术初步研究TRPM8在乳腺癌细胞迁移中的可能作用来探讨TRPM8作为抑制乳腺癌转移的分子靶点的可能性,为乳腺癌早期诊断、治疗评价及判断预后提供参考。
方法
1.检测人体乳腺癌旁组织与乳腺癌组织中TRPM8基因、蛋白水平表达
选取4组配对的乳腺癌旁组织及癌组织标本为研究受试对象。采用QPCR、 Western Blot方法分别在mRNA.蛋白表达水平检测TRPM8在人体乳腺癌旁组织与乳腺癌组织之间的表达量是否存在差异。
2.检测人正常乳腺上皮细胞株与人乳腺癌细胞株TRPM8基因、蛋白水平表达选取正常乳腺上皮细胞株MCF-10A以及不同转移能力乳腺癌细胞株MCF-7、T47D. MDA-MB-231、BT549、SKBR3、ZR-75-30为研究受试对象。采用QPCR、 Western Blot方法分别在mRNA、蛋白表达水平检测TRPM8在正常乳腺上皮细胞株MCF-10A及不同转移能力乳腺癌细胞株中的表达差异。
3.RNAi干扰TRPM8基因对人乳腺癌高转移细胞株MDA-MB-231迁移能力的影响利用基因公司设计合成的3条靶向TRPM8基因的siRNA片段(用TRPM8-RNAi-1、RPM8-RNAi-2和TRPM8-RNAi-3表示)和1条阴性对照RNAi片段(用Scramble表示),分别将4条siRNA片段转染进入不同的MDA-MB-231细胞,利用QPCR和Western Blot方法分别检测各组细胞的]PRPM8表达水平,筛选出TRPM8-RNAi-1-3中干扰效率最高的siRNA片段。接着利用RNA干扰(RNAi)技术干扰高表达TRPM8的高转移能力的MDA-MB-231细胞株其TRPM8mRNA表达量,采用QPCR. Western Blot方法检测TRPM8基因干扰效果,采用三维培养模型观察干扰细胞的形态改变,分别用细胞划痕实验及transwell小室实验检测已成功干扰TRPM8的人乳腺癌细胞MDA-MB-231/siTRPM8的迁移能力变化。
4.稳定转染TRPM8基因对人乳腺癌低转移细胞株MCF-7迁移能力的影响
将TRPM8目的基因序列克隆入pBABE-puro表达载体,建立pBABE-puro-TRPM8表达载体。经限制性内切酶酶切鉴定、测序并转染293FT细胞判断成功构建pBABE-puro-TRPM8表达载体。将空质粒和pBABE-puro-TRPM8表达载体分别转染进入人乳腺癌MCF-7细胞,建立稳定表达TRPM8的MCF-7/pBABE-puro-TRPM8细胞,应用QPCR、Western Blot检测稳定转染细胞的TRPM8基因及蛋白表达水平；采用三维培养模型观察稳定转染TRPM8基因的细胞形态改变,并运用细胞划痕实验、transwell小室实验检测稳定转染TRPM8基因的MCF-7细胞的迁移能力变化。
5.TRPM8影响MCF-7、MDA-MB-231细胞迁移能力机制的初步探讨
选取沉默内源性TRPM8基因的MDA-MB-231细胞及稳定转染TRPM8基因的MCF-7细胞为受试细胞,采用QPCR、Western blot分别在mRNA、蛋白表达水平检测沉默TRPM8前、后MDA-MB-231细胞及转染TRPM8前、后MCF-7细胞中E-cadherin、Vimentin、Fibronectin、AKT、p-AKT、GSK-3β、p-GSK-3β、 Snail的表达变化,以探讨TRPM8基因对人乳腺癌细胞迁移能力的影响及其相关机制。
6.统计学方法：
采用SPSS21.0统计软件进行处理,计量资料用均数±标准差(x±s)表示,若方差齐性采用ONE-WAY ANOVA,多重比较采用LSD法,若方差不齐,整体比较采用Welch检验,多重比较则采用Dunnett's T3检验,以P<0.05为差异有统计学意义；mRNA检测则采用单样本t检验,以P<0.05为差异有统计学意义；两组比较,采用两独立样本t检验,以P<0.05为差异有统计学意义。
结果
1.TRPM8在人体乳腺癌旁组织和乳腺癌组织mRNA、蛋白水平的表达：
4例标本的检测结果均表明人乳腺癌组织中TRPM8在mRNA、蛋白表达水平上均较其在人乳腺癌旁组织中的表达水平高,人体乳腺癌组织和人乳腺癌旁组织其TRPM8mRNA及蛋白表达差异均有统计学意义(P<0.05)。
2.TRPM8在人正常乳腺上皮细胞株与人乳腺癌细胞株mRNA、蛋白水平的表达：
QPCR和Western blot检测正常乳腺上皮细胞株MCF-10A及6种人乳腺癌MCF-7、T47D、MDA-MB-231、BT549、SKBR3、ZR-75-30细胞中TRPM8在mRNA和蛋白水平的相对表达量,两种检测方法的结果基本一致：①在mRNA及蛋白水平,6种人乳腺癌细胞的TRPM8相对表达水平都高于其在正常乳腺上皮细胞的表达水平；②正常乳腺上皮细胞株MCF-10A的TRPM8相对表达水平最低；③高转移能力乳腺癌细胞MDA-MB-231的TRPM8表达水平最高；④低转移能力乳腺癌细胞MCF-7的TRPM8表达水平相对较低；⑤各乳腺癌细胞与正常乳腺上皮细胞株MCF-10A之间分别在TRPM8mRNA、蛋白水平的表达量比较差异有统计学意义(P<0.05)；⑥6株乳腺癌细胞株间的TRPM8mRNA、蛋白相对表达水平多重比较,差异均有统计学意义(P<0.05)。
3.沉默TRPM8基因对人乳腺癌细胞株MDA-MB-231迁移能力的影响：
干扰TRPM8基因效率最高的干扰片段是siRNA3(约80%)；稳转TRPM8RNAi-3的MDA-MB-231细胞其TRPM8mRNA、蛋白表达水平下降,细胞间连接比较紧密,克隆边缘较光滑,未见明显的伪足。
划痕实验结果表明在Oh时MDA-MB-231/Scramble组和MDA-MB-231/siTRPM8组细胞的划痕面积基本相等,在24h时MDA-MB-231/Scramble组的细胞划痕面积小于MDA-MB-231/siTRPM8组细胞划痕面积,在48h时MDA-MB-231/Scramble组的细胞划痕面积小于MDA-MB-231/siTRPM8组细胞划痕面积。随观察时间的推移,两组细胞其无细胞划痕区逐渐变窄,沉默了TRPM8的MDA-MB-231细胞其细胞迁移能力减弱。
Transwell小室实验结果显示MDA-MB-231/siTRPM8的穿孔细胞数少于MDA-MB-231/Scramble的穿孔细胞数,与MDA-MB-231/Scramble细胞比较,MDA-MB-231/siTRPM8细胞的迁移能力降低(t=5.043,P=0.006),实验初步表明沉默TRPM8基因可能抑制了MDA-MB-231细胞的迁移能力。
通过划痕实验和Transwell小室实验得出干扰TRPM8表达后的MDA-MB-231细胞侵袭、迁移能力被抑制,这说明TRPM8可能促进乳腺癌细胞的转移。
4.TRPM8基因过表达对人乳腺癌细胞株MCF-7迁移能力的影响：
稳定转染pBABE-puro-TRPM8表达载体的原来低侵袭能力的MCF-7细胞经QPCR, Western Blot方法检测其TRPM8的表达水平,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,将其命名为MCF-7/TRPM8细胞,而对照组细胞命名为MCF-7/vector细胞。实验结果表明MCF-7/vector组与MCF-7/TRPM8组细胞其TRPM8mRNA、蛋白表达水平差异有统计学意义(P<0.05),结果提示TRPM8表达水平在MCF-7/TRPM8细胞呈上调,初步证实MCF-7/TRPM8细胞已成功转染TRPM8基因。
在细胞三维培养模型中,MCF-7/TRPM8细胞可见克隆伸出伪足向四周浸润,边界不整齐,细胞间连接较松散,从其细胞形态学改变提示乳腺癌稳定转染TRPM8基因的细胞株MCF-7/TRPM8的侵袭能力增强。
划痕实验结果表明在24h时、48小时MCF-7/TRPM8组的细胞划痕面积均小于MCF-7/vector组细胞划痕面积,随观察时间的推移,两组细胞无细胞划痕区逐渐变窄,稳定转染TRPM8的MCF-7细胞其细胞迁移能力增强。
Transwell小室实验结果显示MCF/TRPM8的穿孔细胞数高于MCF-7/Vector的穿孔细胞数,其差异有统计学意义(t=-13.058,P<0.001),实验初步表明稳定转染TRPM8基因后可能增强了MCF-7细胞的迁移能力。
5.TRPM8的表达高、低对人乳腺癌细胞株MCF-7、MDA-MB-231迁移影响的作用机制：
本实验结果表明在RNA和蛋白水平上,均显示稳定转染TRPM8的MCF-7细胞,其上皮标志物E-cadherin的表达量下调,而Vimentin、Fibronectin、Snail的表达量上调；免疫荧光实验提示乳腺癌MCF-7/Vector细胞的细胞膜上高表达E-cadherin蛋白,而其胞浆内Vimentin蛋白呈低表达；而乳腺癌MCF-7/TRPM8细胞的细胞膜上E-cadherin蛋白呈低表达,其细胞胞浆内、Vimentin蛋白则呈高表达。
在mRNA和蛋白水平上,MDA-MB-231/siTRPM8细胞的上皮标志物E-cadherin的表达量均增强,而其Vimentin、Fibronectin、Snail表达均不同程度减弱；免疫荧光实验提示乳腺癌MDA-MB-231/siTRPM8细胞的细胞膜上E-cadherin蛋白呈高表达,其胞浆内Vimentin蛋白则呈低表达；而乳腺癌MDA-MB-231/Scramble细胞的细胞膜上E-cadherin蛋白呈低表达,而其胞浆内Vimentin蛋白呈高表达。
在MDA-MB-231/siTRPM8细胞,其Akt、GSK-3β的mRNA、蛋白表达均无改变,但其Akt的磷酸化水平下降,随着p-AKT蛋白表达量的减少,GSK-3β的磷酸化水平亦呈下降趋势,P-GSK-3β蛋白表达量减少；而在稳定转染TRPM8基因的MCF-7细胞中AKt及GSK-3βmRNA、蛋白亦无改变,但其Akt的磷酸化水平(p-AKT)蛋白表达量增加,下游靶蛋白GSK-3β的磷酸化水平亦同步呈上升趋势,即P-GSK-3β蛋白表达量升高。
结论
1.人乳腺癌组织中存在TRPM8过表达的现象；
2.乳腺癌细胞中TRPM8的mRNA及蛋白相对表达量的高低与其细胞侵袭、转移能力可能有关,即细胞中的TRPM8相对表达量越高,其侵袭、转移能力越强；
3.沉默TRPM8基因的MDA-MB-231细胞(MDA-MB-231/siTRPM8)侵袭、迁移能力被抑制,这说明TRPM8可能促进乳腺癌细胞的转移；
4.细胞TRPM8表达水平的高低可能是细胞迁移能力强弱的标志,TRPM8基因过表达可能增强MCF-7细胞的迁移能力,乳腺癌细胞中TRPM8表达量越高,其细胞迁移能力越强,TRPM8可能是乳腺癌的一个转移相关基因；
5.稳定转染TRPM8基因,可能会增强低侵袭性乳腺癌MCF-7细胞发生EMT现象而增强肿瘤细胞的侵袭、转移能力,而沉默TRPM8基因可能会抑制高侵袭性乳腺癌MDA-MB-231细胞发生EMT现象而抑制肿瘤细胞的侵袭、转移能力,TRPM8增强肿瘤细胞的侵袭、转移能力的作用机制可能与其通过Akt/GSK-3p信号转导通路参与EMT过程有关。
本研究的创新之处
1.验证了乳腺癌组织中存在TRPM8过表达现象；
2.利用重组质粒pSuper-retro-puro-siTRPM8转染MDA-MB-231细胞株,筛选出稳定沉默TRPM8基因的MDA-MB-231/siTRPM8细胞株；成功构建pBABE-puro-TRPM8表达载体,转染人乳腺癌MCF-7细胞,建立了TRPM8基因过表达的稳定人乳腺癌细胞株MCF-7/TRPM8,为研究TRPM8基因的功能提供了有价值的研究工具。
3.发现TRPM8可能与乳腺癌细胞迁移相关,为开展乳腺癌基因靶向治疗初步奠定了理论基础。
4.首次阐明了TRPM8可能通过Akt/GSK-3β信号通路影响乳腺癌细胞EMT从而影响肿瘤细胞的迁移。
Malignant tumor is common chronic disease, which has become a significant public health problems worldwide. Research shows that malignant tumor incidence rate of285.91/10million at2009in China, the malignant tumor mortality rate was180.54/10million. Breast cancer is the most common form of cancer in women in western countries, and already taking the first place of incidence in chinese female cancer patients at2009. Metastatic breast cancer is one of the most common malignant tumors, there are about one million two hundred thousand women with breast cancer around the world every year, half of the patients died within10years.
Methods in the treatment of malignant tumor include surgical operation, radiotherapy, chemotherapy, biologic therapy, interventional therapy, radiofrequency therapy, cryotherapy, laser therapy and Chinese medicine treatment, but the drug resistance and metastasis of tumor cells are still the main factors that hinder the effectiveness of treatment of malignant tumor. Invasion and metastasis is the important cause of death in patients with malignant tumor, more than90%of the patients died of the tumor metastasis or recurrence, routine treatments cannot fundamentally eliminate malignant tumor. Nowadays the basic and clinical researches of tumor focus on the mechanism of malignant tumor invasion and metastasis.
Tumor invasion and metastasis process is very complex, even though different kinds of tumors invasion and metastasis have their own different characteristics. The key steps are basically the same in the process of invasion and metastasis of tumor.
In the last10years, the extent to which TRP channels are associated with cancer has been increasingly clarified, and the involvement of these receptors in triggering enhanced proliferation, aberrant differentiation, and impaired ability to die, thus leading to the uncontrolled cancer expansion and tumor invasion have been recognized. More and more scholars work on the TRP channel in the changes of physiological function and cancer.
TRPM channel is a non-selective cation channel superfamily, TRPM8belongs to the TRPM (melastatin) subfamily of TRP proteins. TRPM8is widely distributed, it plays an important role in the occurrence and development of some tumors. Studies had shown that TRPM7was related with the invasion and metastasis of breast cancer, also some researches had indicated that over expression of TRPM8was observed in human breast cancer tissues, and TRPM8has become a clinical index of the bad prognosis of prostate cancer patients, study on the mechanism of TRPM8will provide clues for the study of tumor.
This study attempted to explore the possibility of TRPM8as a molecular target in inhibiting metastasis of breast cancer, TRPM8channel might represent valuable diagnostic and/or prognostic markers, as well as targets for pharmaceutical intervention and targeting in cancer.
Methods
1. Tissue samples
Primary cancer tissues and adjacent non-tumor tissues were obtained from surgical treatments of breast cancer. All samples were formalin-fixed and paraffin-embedded (FFPE) with standard procedures. The histological diagnosis was made by a pathologist and has been re-confirmed by a second pathologist. The study was approved by the Institutional Review Board (IRB) in the Southern Medical University and consented by patients involved.
2. Cell culture
Breast epithelial cell line MCF-10A and metastatic breast cancer cell lines including MCF-7, T47D, MDA-MB-231, BT549, SKBR3and ZR-75-30were purchased from American Type Culture Collection (ATCC) and preserved in Central Laboratory in Southern Medical University. Each cell line is maintained in culture medium and atmosphere instructed by each manuscripts.
3. Generation of stable cell lines
The sequence of TRPM8and shRNA-TRPM8were cloned into pMSCV-puromycin and pSuper-puromycin retroviral vectors respectively, and the resulting plasmid was transfected into293FT cells to generate virus as described previously. After incubation at37℃for6h, the transfected cells were cultured in fresh media overnight. In the following days, media were collected three times a day to gather the produced virus until the293FT cells reached total confluency. Media containing pMSCV/TRPM8or pSuper/shRNA-TRPM8were used to infect MCF-7or MDA-MB-231respectively for24h. After removal of the inoculum and replacement with fresh media, infected cells were selected by adding puromycin. Stable cell lines were verified by Western blotting and qRT-PCR.
4. Quantitative Real Time-PCR (qRT-PCR) analysis
Total RNA from different cell lines and human tissues was extracted using Trizol reagent. cDNA was synthesized from2.5μg of total RNA using random hexamers. Real-time PCR was carried out using an ABI PRISM7500Sequence Detection System. Reactions were run in triplicate in three independent experiments.The geometric mean of the housekeeping gene β-actin was used as internal control to normalize the variability in expression levels.
5. Wound healing assay
One day before scratch, MCF-7/TRPM8and MDA-MB-231/RNAi cells were trypsinized and seeded equally into6-well tissue culture plates, and grew to reach almost total confluence in24h. When non-serum starvation kept for24h after cell monolayer formed, an artificial homogenous wound was created onto the monolayer with a sterile200μL tip. After scratching, the cells were washed with serum-free medium. Images of cells migrating into the wound were captured at time points of0h,24h and48h by inverted microscope (200×).
6. In vitro invasion assay
The invasion assay was done by using Transwell chamber consisting of8mm membrane filter inserts coated with Matrigel as previously described. Briefly, cells were trypsinized and suspended in serum-free medium. Then1×104cells were added to the upper chamber, whereas lower chamber was filled with medium with10%FBS. After incubated for48h, cells were invaded through the coated membrane to the lower surface, in which cells were fixed with4%paraformaldehyde and stained with hematoxylin. The cell count was done under the microscope (200×).
7. Western blotting
For the expression analysis of EMT-related proteins, immunoblotting assay was carried out. MCF-7/TRPM8and MDA-MB-231/RNAi cells were seeded in100mm tissue culture dishes. After24h, cells were washed with prechilled PBS when the confluence reached to60-70%, followed by being harvested in sample buffer [62.5mmol/L Tris-HCl (pH6.8),2%SDS,10%glycerol, and5%2-b-mercaptoethanol]. Equal amounts of protein from the supernatant were loaded per lane and resolved by SDS-polyacrylamide electrophoresis. In sequence, protein was transferred onto PVDF membrane, blocked by5%nonfat milk for1h at room temperature, and probed with primary antibodies for3h. Primary antibodies for E-Cadherin, Fibronectin, Vimentin and Snail were purchased from BD Biosciences. Those for AKT, p-AKT, GSK-3β p-GSK-3β were purchased from Cell Signaling Technology. Membranes were washed thrice (10min each) in TBS-T buffer and incubated for40min at room temperature with horseradish peroxidase-conjugated anti-mouse secondary antibodies. Blots were washed thrice (10min each) in TBS-T and developed using the ECL system. Protein loading was normalized by reprobing the blots with a-Tubulin antibody.
8. Immunofluorescence analysis
Cells were stained for immunofluorescence on coverslips as described previously. Briefly, the cells were incubated with primary antibodies against E-cadherin and Vimentin, and then incubated with rhodamine-conjugated or FITC-conjugated goat antibodies against rabbit or mouse IgG. The coverslips were counterstained with DAPI and imaged with a confocal laser-scanning microscope.
9.3D morphogenesis assay
Twenty-four-well dishes were coated with Growth Factor Reduced Matrigel and covered with growth medium supplemented with2%Matrigel as previously described. Cells were trypsinized and seeded at a density of104cells/well, and medium was replaced with2%Matrigel for3to4days. Microscopic images were captured at2-day intervals for2-3weeks.
10. Statistical analysis
Statistics were assessed using SPSS21.0(SPSS, Inc., Chicago, IL, USA). Statistical data are expressed as mean±SEM, When appropriate, Student's t-test and one-way ANOVA were applied. In all cases, P<0.05was considered statistically significant. All experiments were repeated at least three times.
Results
1. The expressions of TRPM8in human breast adjacent non-tumor tissues and tumor tissues.
Four pairs of resected breast tissues were included to our study. TRPM8protein levels were obviously higher in tumor tissues than in the pairing adjacent non-tumor tissues. To consolidate this finding, we detected mRNA expressions of TRPM8in each sample pairs, and result is consistent with of protein level.
2. The expressions of TRPM8in one human breast normal epithelial cell and in six human breast cancer cells.
Expression of TRPM8was examined at transcription and protein level in one breast normal epithelial cell line and in six cancer cell lines using western blotting and qRT-PCR. A very strong to moderate expression of TRPM8was observed in all cell lines except for MCF-10A cell that showed very low expression which was normalized as base line in qRT-PCR. Among the six breast cancer cell lines, MDA-MB-231showed the highest expression of TRPM8, while MCF-7, T47D and ZR-75-30demonstrated relatively lower TRPM8levels
3. Effects of TRPM8gene silencing on the migration of human breast cancer cell line MDA-MB-231
We interfered endogenous expression of TRPM8in MDA-MB-231by transfecting silencing plasmids, expression of TRPM8decreased at transcription and protein level in MDA-MB-231/siTRPM8. Morphologically, low expression of TRPM8induced major3D-shape changes in comparison to control cells, The cells connections of MDA-MB-231/siTRPM8were closer, the edge of MDA-MB-231/siTRPM8was smoother, there was not obvious pseudopodia in MDA-MB-231/siTRPM8.
Cell migration observed by wound healing assay, cells transfected with siTRPM8moved more slowly than those with scramble sequence.The results of Transwell chamber experiments showed that MDA-MB-231/siTRPM8cells penetrated through the gel-membrane section significantly less than MDA-MB-231/Scramble, The migration ability of MDA-MB-231/siTRPM8cells decreased significantly(t=5.043, P=0.006), The preliminary results showed that TRPM8gene silencing could inhibit the migration of MDA-MB-231cells.
4. Effects of TRPM8gene over-expression on the migration of human breast cancer MCF-7cell
To investigate the role of TRPM8in the development and progression of breast cancer, TRPM8over-expression cells were established by plasmid transfection to perform migration and invasion assays in vitro. The experimental results showed that TRPM8expression level was upregulated in MCF-7/TRPM8cells, which confirmed that MCF-7/TRPM8cells have been successfully transfected with TRPM8gene.
As we expect, cell migration observed by wound healing assay demonstrated that cells transfected with TRPM8plasmid significantly ran faster than those with vector control.
Three dimensional cell culture model showed MCF-7/TRPM8with the formation of thorns or pseudopodia invaded the surrounding, which suggested MCF-7/TRPM8had more motile and higher invasiveness. Cell migration observed by wound healing assay, MCF-7cells transfected with TRPM8moved more faster than those with empty vector.
The invasive property of MCF-7cells was examined by Transwell-Matrigel penetration assay, which depicted cells transfected with TRPM8penetrated through the gel-membrane section much more than cells transfected with vector(MCF-7/Vector). The preliminary results showed that stable transfection of TRPM8gene could enhance the migration ability of MCF-7cells.
5. The mechanism of TRPM8how to effect on the migration of MCF-7and MDA-MB-231
To investigate whether TRPM8regulates malignancy and metastatic features of breast cancer cells by promoting EMT, we interfered endogenous expression of TRPM8in MDA-MB-231by transfecting silencing plasmids while increased TRPM8level in MCF-7by introducing TRPM8-expressing constructs, over-expression of TRPM8repressed E-cadherin, one of epithelial markers and supposed to be down-regulated during EMT. Nevertheless, E-cadherin was increased in breast cancer cells transfected with TRPM8silencing plasmids. Moreover, Fibronectin and Vimentin, which are mesenchymal markers and should be up-regulated during EMT, were repressed in TRPM8-silenced cells. However, they were significantly increased in cells transfected with TRPM8plasmid. In order to observe visually, we probed the cells by immunofluorescent staining. cells with high TRPM8exhibited obvious down-regulation of E-cadherin and up-regulation of Vimentin, vice versa. These suggested that TRPM8had positive effects on EMT in breast cancer and might conduct epithelial cells to transform toward mesenchymal cells.
Changing TRPM8expression did not obviously influence the expression levels of Akt and GSK-3β, but phosphorylation of Akt was significantly inhibited in cells transduced with siRNA against TRPM8(MDA-MB-231/siTRPM8). The increase in Akt phosphorylation was accompanied by a change in phosphorylation of GSK-3P, a downstream target protein of Akt, suggesting that up-regulation of TRPM8activates the Akt/GSK-3β pathway in breast cancer cells. Western blotting revealed that, compared with vector cells, expression of Snail significantly increased in breast cancer cells transfected with TRPM8plasmid but decreased in those with silencing constructs.
Conclusions
1. TRPM8was highly expressed in human breast cancer tissues.
2. TRPM8was a key gene/protein related to migration of human breast cancer, increasing the expression of TRPM8would enhance the migration of breast cancer cells.
3. TRPM8gene silencing inhibited the invasion and migration of MDA-MB-231, which indicated TRPM8might promote the migration of breast cancer cell.
4. TRPM8may be a metastatic related gene in breast cancer.
5.TRPM8enhances the invasion and migration of tumor cells, whose mechanism may be involved in EMT process by the Akt/GSK-3β signaling pathway.
Innovations of our study
1. This study has verified that over-expression of TRPM8exists in breast cancer tissues.
2. Stable silencing TRPM8cell line MDA-MB-231/siTRPM8was established with pSuper-retro-puro-siTRPM8transfection and puro selection, and stable TRPM8-transduced breast cancer line MCF-7was established with pBABE-puro-TRPM8, which would be provided a valuable tool for TRPM8functional studies.
3. TRPM8could be related to the migration of human breast cancer, which would provide theoretical basis on targeted therapy of breast cancer.
4. The preliminary study demonstrated how TRPM8channels function to regulate the migration of breast cancer cells by Akt/GSK-3β pathway which affected human breast cancer EMT.
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[8][8] Santoni G, Farfariello V, Amantini C. TRPV channels in tumor growth and progression[J[. Adv Exp Med Biol,2011,704:947-967.
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