Tiam1基因鼻咽癌侵袭转移中的作用及其相关信号通路的初步研究
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摘要
研究背景和目的
鼻咽癌(nasopharyngeal carcinoma,NPC)是我国南方及东南亚地区常见的一种恶性肿瘤,尤以广东省珠江三角洲一带多见,俗有“广东癌”之称。虽然近年来鼻咽癌的诊断和治疗水平已有很大提高,但鼻咽癌的5年生存率仍很低,究其原因,转移是导致鼻咽癌患者治疗效果和死亡的关键。随着分子生物学研究的进展,尤其是基因芯片技术的应用,目前已经探索出许多与鼻咽癌转移有关的分子标志物,如VEGF、CXCR4、CD44、Cadherin、MMP、nm-23、Wt-p53等,它们在鼻咽癌侵袭转移中均发挥重要作用。有关T淋巴瘤侵袭转移诱导因子1(T lymphoma invasion and metastasis inducing factor 1,Tiam1)基因在鼻咽癌侵袭转移中的功能及机制研究目前少有报道。
Tiam1基因是采用前病毒插入致突变的原理,在BW5147小鼠T淋巴瘤细胞高侵袭变异株中分离得到,与肿瘤生物学特性的关系已逐步成为目前的研究热点。研究表明,Tiam1在乳腺癌、肺癌、结直肠癌等多种肿瘤组织中表达,特别在低分化的和伴有局部浸润或远处转移的腺癌、鳞癌组织中呈强阳性表达。通过对来源于人和啮齿类肿瘤源性的40多个细胞系的检测发现,Tiam1在大多数肿瘤细胞中表达,但在来源于相同组织类型的不同肿瘤细胞系中Tiam1的表达依据其肿瘤侵袭转移能力的不同而异。另有研究认为,Tiam1基因过表达可促进结直肠癌细胞的侵袭转移作用,Tiam1基因沉默可降低结直肠癌细胞的侵袭转移能力。也有研究发现,Tiam1基因敲除的小鼠能够抵抗诱导的皮肤癌发生,即使产生肿瘤,其数量、生长速度及转移瘤发生率也远较野生型荷瘤鼠低。Tiam1诱导肿瘤细胞侵袭转移的分子机制与肿瘤细胞骨架、肿瘤细胞粘附分子、肿瘤细胞膜皱褶、片状伪足形成以及细胞外基质密不可分。还有研究表明,Ankyrin蛋白以其ADR(ankyrin repeat domain)区与Tiam1结合,通过Ankyrin-Tiam1-Rac1实现细胞骨架结构重塑,促进乳腺癌细胞的侵袭转移;癌基因CD44-V3可与Tiam1蛋白的PHn-CC-Ex功能域特异性结合,从而调节Tiam1-Rac信号,促进乳腺癌SP1细胞的侵袭转移。
浸润转移是鼻咽癌最重要的生物学特性之一,尽管Tiam1基因在多种肿瘤组织及肿瘤细胞系中均有表达且与肿瘤转移的关系已基本明确,但在人鼻咽癌中的表达、作用及机制目前国内少有文献报道,国外未见报道。因此,本研究结合临床资料,应用基因转染、基因沉默及体内外实验等研究方法,从人群和分子水平探讨Tiam1基因在鼻咽癌发生发展中的作用及其机制,为鼻咽癌转移的个体化基因治疗提供新靶标和新方法。
方法
1.Tiam1在鼻咽癌中的表达及其作用
应用免疫组化SP-9000法检测71例鼻咽未分化型非角化性癌、20例慢性鼻咽粘膜炎、6例人鼻咽癌细胞株裸鼠转移模型肿瘤组织中Tiam1蛋白的表达,同时运用免疫荧光和RT-PCR方法分别检测Tiam1在6种鼻咽癌细胞株中的表达。
2.Tiam1过表达对鼻咽癌细胞株侵袭转移特性的影响
根据Tiam1基因在6种人鼻咽癌细胞株中的表达情况,选择Tiam1中度表达、具有一定成瘤及转移能力的细胞株,利用阳离子脂质体LipofectamineTM2000法将Tiam1/C1199 cDNA质粒导入人鼻咽癌细胞,G418筛选抗性单克隆后扩大培养,用RT-PCR及Western blot方法鉴定转染后Tiam1基因在细胞中的表达情况并进行体内外功能验证实验。
3.Tiam1基因沉默对鼻咽癌细胞侵袭转移特性的影响
针对Tiam1(NM 003253)靶基因序列,利用公用网站、按照RNA干扰序列设计原则,设计A、B、C、D四个RNA干扰靶点序列,将含各干扰序列的双链DNA oligo粘性两端酶切(AgeI/EcoRI酶切)并连接到酶切后线性化的RNA干扰载体PGC-LV-GFP上。将连接好的产物转入制备好的细菌感受态细胞,对长出的克隆先进行菌落PCR鉴定,再进行测序比对,鉴定后的阳性克隆即为构建成功的目的基因RNA干扰慢病毒载体(即VshRNA)。将含各干扰序列的VshRNA质粒与pHelper1.0和pHelper2.0载体质粒共转染293T病毒包装细胞,收集富含慢病毒颗粒的细胞上清液,对其浓缩后得到高滴度的慢病毒浓缩液,在293T细胞中测定并标定病毒滴度。分别用含Tiam1各干扰序列的慢病毒感染人鼻咽癌细胞,同时设置阴性对照。用Real-time PCR方法初步筛选有效的干扰靶点,根据Real-time PCR初步筛选结果,选择干扰效果较好的两种病毒液再次感染CNE2细胞,待感染时间达到7天后收集细胞,进行荧光定量PCR和Westernblot鉴定。选取最有效的病毒液感染的鼻咽癌细胞用于后续的体内、体外功能验证实验。
4.Tiam1相关信号通路的初步探讨
利用基因通路网站(http://www.genome.jp/kegg/)、酵母双杂交网站(http://visant.bu.edu/)等得到若干与Tiam1相互作用的蛋白,从中选取与Tiam1侵袭转移特性密切相关的Ankyrin1、Rac1、CD44蛋白进行研究。运用免疫荧光双标记技术验证人鼻咽癌细胞中Tiam1-Ankyrin1、Tiam1-Rac1、Tiam1-CD44的表达并进行共定位分析;采用免疫共沉淀、免疫印迹技术验证Tiam1与CD44、Ankyrin1、Rac1蛋白的相互作用,探讨Tiam1在鼻咽癌侵袭转移中参与相关信号通路及其与CD44、Ankyrin1、Rac1蛋白的相互调控关系。
结果
1.鼻咽癌组织、鼻咽癌细胞株中Tiam1的表达
免疫组化结果表明,Tiam1蛋白在鼻咽癌组中的阳性表达得分为2.943±1.297,显著高于慢性炎症组0.700±0.923(t=7.224,P＜0.01);鼻咽癌组Tiam1蛋白表达得分与年龄无相关关系(r=0.020,P=0.871),其得分在性别之间、在T分期之间也没有统计学差异(t=1.702,P=0.093;F=1.670,P=0.182),而在有无淋巴结转移组间和有无远处器官转移组间差异具有统计学意义(t=9.005,P＜0.01;t=5.069,P＜0.01)。在具有转移特性的人源性鼻咽癌细胞株裸鼠模型肿瘤组织中Tiam1蛋白均呈高表达;运用免疫荧光和RT-PCR方法检测Tiam1在6种人鼻咽癌细胞株中的表达时发现,Tiam1在鼻咽癌高转移细胞株5-8F中表达(0.560±0.020)高于无转移特性的细胞株6-10B中的表达(0.110±0.010)(t=34.86,P＜0.01)。以上研究结果表明Tiam1与鼻咽癌的侵袭转移特性相关。
2.Tiam1稳定转染人鼻咽癌细胞株的建立及其侵袭转移特性的变化经RT-PCR及Western blot方法验证显示各转染克隆细胞中Tiam1的表达均高于未转染或空载体转染的鼻咽癌细胞,表明成功建立了Tiam1过表达的鼻咽癌细胞株。选取Tiam1表达最强的细胞克隆分别进行以下的体内外功能验证实验。
MTT实验结果证实,在细胞培养的第1天,转染了Tiam1的CNE2细胞的增殖能力与空载体转染组和未转染组比较没有统计学差异(F=0.740,P=0.498);随着细胞培养天数的增加,CNE2/Tiam1组细胞的增殖能力明显超过前两组(第2-7天各细胞组单因素方差分析的F值分别为3.289、15.519、3.616、58.265、28.983、57.459,P值分别为0.073、0.000、0.059、0.000、0.000、0.000),表明Tiam1过表达增强了鼻咽癌CNE2细胞的体外增殖能力。
平板克隆形成实验、软琼脂集落形成实验结果均显示,Tiam1过表达的CNE2细胞较未转染细胞其克隆生长能力增加,差异具有统计学意义(平板克隆形成实验:t=25.065,P＜0.01;软琼脂集落形成实验:F=318.960,P＜0.01)。
粘附实验结果表明,Tiam1基因过表达的CNE2细胞对FN的粘附能力高于未转染细胞和空载体转染细胞,差异具有统计学意义(F=86.689,P＜0.05)。
划痕试验检测Tiam1基因转染后对人鼻咽癌细胞株CNE2迁移能力的影响。方差分析结果是:Tiam1过表达增加了鼻咽癌细胞的迁移能力(F=356.753,P＜0.05)。
细胞侵袭实验证明,Tiam1转染细胞与空载体转染、未转染细胞相比较,其穿过基底膜的细胞数显著增多,差异具有统计学意义(F=323.158,P＜0.05)。
鼻咽癌裸鼠体内转移实验发现,在Tiam1转染组的8只裸鼠肺内均形成了明确的转移灶,在空载体转染组的9只裸鼠体内有4只形成肺内转移灶。Tiam1转染组每只裸鼠肺内所形成的转移灶个数(7.75±2.315)明显大于空载体转染组(3.78±4.549)(t′=2.305,P＜0.05)。其中在1只Tiam1过表达组的裸鼠肝组织内还可见散在的鼻咽癌细胞浸润。
3.Tiam1稳定沉默鼻咽癌细胞株的建立及其侵袭转移特性的改变
将含Tiam1干扰序列A、B、C、D的病毒液以及阴性对照的病毒液分别感染人鼻咽癌CNE2细胞,7天后收集细胞经过Real-time PCR的初步筛选得到,B序列干扰效果最好(0.124±0.055),C序列次之(0.156±0.045);A和D序列的干扰效果相对较低(A,0.230±0.043;D,0.255±0.116)。与未感染组和阴性对照组比较,差异具有统计学意义(F=69.732,P＜0.05)。为了确保结果的可靠性,又将含B、C干扰序列的慢病毒分别和混合感染人鼻咽癌CNE2细胞,培养7天后收集细胞再次进行Real-time PCR鉴定及Western blot鉴定,两种验证方法所得出的结果与初步筛选时所得出的结论相一致;并将感染B慢病毒的CNE2细胞命名为KD,感染阴性对照慢病毒的CNE2细胞命名NC,未感染的CNE2细胞命名CON,并进行以下的体内、体外功能验证实验。
采用流式细胞术分析三组细胞的细胞周期分布,结果发现Tiam1/KD组的CNE2细胞处于G1期的比例增高,处于S期和G2+M期细胞的比例则相应降低,与NC及CON组比较,差异具有统计学意义(F值分别为2021.007、1141.527、73.162,P＜0.05),表明Tiam1干扰后细胞的增殖能力下降。
软琼脂集落形成实验结果表明Tiam1/KD细胞其单个细胞的增殖能力较未干扰的鼻咽癌细胞降低,差异具有统计学意义(F=645.863,P＜0.05)。
异质粘附实验结果发现,Tiam1/KD细胞对FN的异质粘附能力下降,与未干扰的CNE2细胞比较,差异具有统计学意义(F=55.388,P＜0.05)。
采用细胞侵袭实验分析Tiam1干扰后细胞体外侵袭能力的变化,统计分析结果显示Tiam1/KD细胞穿过人工基底膜胶的侵袭细胞数量显著低于Tiam1未干扰的CNE2细胞(F=181.727,P＜0.05),表明Tiam1干扰后的CNE2细胞的体外侵袭能力降低。
运用考马斯亮蓝细胞骨架染色方法观察Tiam1干扰前后细胞骨架的含量及分布变化,结果表明Tiam1/KD组的CNE2细胞其细胞骨架密集,粗大,沿细胞形态走向排列,可见明显的细胞突起;而NC、CON组的未干扰细胞的细胞骨架呈稀疏、散在分布。
扫描电镜结果显示NC、CON组细胞与玻片贴附紧密,微绒毛丰富,相互间形成连接;而Tiam1干扰后的CNE2细胞则细胞表面微绒毛稀疏、短小,可见中断现象,进一步印证了细胞骨架染色的结果。
透射电镜观察Tiam1干扰前后细胞超微结构的变化,发现NC、CON组的CNE2细胞,其细胞核内染色质淡染、分布均匀,线粒体、内质网及高尔基复合体(Golgi complex)等细胞器结构完整正常;而Tiam1干扰后的KD细胞其核内异染色质增多,常染色质减少;胞浆内可见裂隙及囊状空泡,可见细胞凋亡小体形成。
采用尾静脉注射法建立了Tiam1沉默前后鼻咽癌细胞的裸鼠体内转移模型。2月后处死裸鼠并在整体荧光体视镜下观察发现在NC组的9只裸鼠中有5只出现了肺转移,共有36个肺转移结节,平均每只裸鼠肺转移结节数量为(4.00±4.093);在CNE2/KD组的9只裸鼠中仅有1只可观察到针尖大小的疑似肺内转移结节,差异具有统计学意义(t′=2.841,P＜0.05)。对各组裸鼠的其它器官组织切片观察显示两组裸鼠均未发现鼻咽癌细胞的转移。
4.鼻咽癌中Tiam1相互作用蛋白的验证
运用免疫荧光双标记技术验证人鼻咽癌CNE2细胞中Tiam1-Ankyrin1、Tiam1-Rac1、Tiam1-CD44的表达并进行共定位分析。结果显示Tiam1和Ankyrin1蛋白均在胞浆中分布,可见两种蛋白散点状共定位的棕黄色区域;Rac1蛋白在CNE2细胞中丰富表达,与Tiam1蛋白明显共定位于胞浆而呈现明亮的黄色;而在Tiam1-CD44实验组,Tiam1仅和CD44蛋白的胞内区域共定位,在细胞膜内侧面呈现较弱的线型黄色区域,在细胞膜的外侧面及胞膜区域则呈现CD44蛋白标记的绿色荧光。
免疫共沉淀实验进一步验证Tiam1与Ankyrin1、Rac1和CD44蛋白的相互作用,结果只检测到Tiam1与Ankyrin1、Rac1的共沉淀现象,没有检测到Tiam1与CD44蛋白共沉淀的发生。
结论
1.Tiam1基因与鼻咽癌进展密切相关,是促进鼻咽癌增殖、粘附、侵袭及转移的重要基因。
2.Tiam1、Ankyrin1和Rac1三种蛋白相互作用,可能共同参与并促进鼻咽癌侵袭转移的发生,Tiam1可能是鼻咽癌侵袭转移相关信号通路中的关键蛋白之本研究的创新之处
1.采用绿色荧光蛋白(GFP)标记的慢病毒载体和RNAi技术建立了稳定沉默Tiam1蛋白的鼻咽癌细胞系,为进一步研究Tiam 1蛋白在鼻咽癌侵袭转移中的功能和作用提供一个理想的实验平台。
2.利用免疫荧光双标记技术和免疫共沉淀方法对筛选出的Tiam1相互作用蛋白在鼻咽癌细胞中进行共定位分析,初步探讨了Tiam1在鼻咽癌侵袭转移中的相关信号通路。
3.较系统地探讨了Tiam1基因在鼻咽癌中的表达及其与鼻咽癌细胞增殖、粘附与侵袭转移的关系。
BACKGROUND & OBJECTIVE
Nasopharyngeal carcinoma(NPC) is a type of cancer common in Southern China and Southeast Asia,especially those of Cantonese origin.Although the levels of diagnosis and the treatment on NPC have being evidently improved in recent years, the survival rate of 5 years for patients with NPC is still very low,which the main cause of higher mortality is early lymph node and distant organ metastases.Recently, many molecular markers relating invasion and metastasis of NPC have been found such as VEGF,CXCR4,CD44,Cadherin,MMP,nm-23,Wt-p53,etc.They play an important role in NPC's invasion and metastasis.However,the function and mechanism of Tiam1(T lymphoma invasion and metastasis inducing factor 1)in NPC's invasion and metastasis are still unclear.
Tiam1 was identified in 1994 by proviral tagging in combination with in vitro selection for invasiveness from murine lymphoma cells.It had been reported that Tiam1 was extensively expressed in a variety of cancers,such as breast cancer,lung cancer,colorectal cancer,and so on,especially in adenocarcinoma and squamous cell cancer with local infiltration and/or distant metastasis.Moreover,examination on more than 40 rodent and human tumor cell lines demonstrated that Tiam1 expression in virtually all lines.Furthermore,the level of Tiam1 was positively correlated with metastatic potential of tumor cell lines.Another research found that Tiam1 overexpression promoted progression of colorectal cancer cell lines,while downregulating Tiam1 protein inhibited their bionomics such as the invasion and metastasis.Tiam1~(-/-) mice were resistant to the development of Ras-induced skin tumors.
Considerable work had demonstrated that the molecular mechanisms of Tiam1-induced invasion and metastasis were closely correlated to cystoskeleton rearrangement,cellular adhesion,cell cycle progression,gene transcription,formation of cell membrane ruffles,etc.Several recent studies had demonstrated that Tiaml binded to the cytoskeletal protein ankyrin,causing Tiam1-mediated Rac1 activation as well as an increase in breast tumor cell migration and invasion.Similarly,the cellular adhesion molecule CD44 binded to Tiam1 at the PHn-CC-Ex region in SP1 murine breast carcinoma cells.Furthermore,the binding of HA to CD44v3 causes an increase in Tiam1-mediated Rac activation and promoted cytoskeleton-mediated tumor cell migration.
Invasion and metastasis are the main bionomics and directly cause of invalid treatment or death of NPC.Up to now,few reports was found about function and mechanism of Tiam1 in NPC.In this study,we mainly focus on the functional alterations of Tiam1 in NPC tissues and cell lines by gene transfection,RNAi,etc.The aim is insighting into the role of Tiam1 in invasion and metastasis,and providing a molecular marker for NPC's treatment and prognosis.
METHODS
1.Expression of Tiam1 and it's relationship with biological behaviour of NPC.
Immunohistochemistry(SP-9000) was performed to examine the expressions of Tiam1 protein in 71 specimens of NPC tissue,20 specimens of chronic nasopharyngitis tissue,and 6 specimens of tumor tissues from nude mice inoculated with metastatic human NPC cells.The expression of Tiam1 in 6 NPC cell lines was detected in protein level by immunofluorescence staining and in mRNA level by RT-PCR respectively.
2.Establishment and functional verification of Tiam1-overexpressed NPC Cells.
We firstly established stably Tiam1-overexpressed CNE2 cells by transfecting Tiam1/c1199 cDNA with lipofectamin2000.The Tiaml-overexpressed CNE2 clones was screened with G418 and verified by RT-PCR and Western blot.The functional alterations of the resulting transfectants were performed by MTT assay,plate colony formation assay,soft agar assay,cell adhesion assay,cell wounding heal assay, invasion assays in vitro and metastasis assay in vivo through the lateral tail-vein injection.
3.Establishment of Tiam1-knockdown NPC cells and functional alterations in vitro and in vivo.
Lentiviral expression vectors containing enhanced green fluorescence protein (GFP) and Tiam1 small interfering RNA(Lenti-Tiam1si),or the control siRNA (Lenti-NC) gene were constructed.A human monocyte-derived cell line CNE2 was transfected with a different multiplicity of infection(MOI) of Lenti-Tiam1-si-A,B,C, D or Lenti-NC,and cultured to obtain stably-transfected CNE2-KD and CNE2-NC cells.The expression of Tiam1 mRNA was determined by real-time PCR,and Tiam1 protein was detected by western-blot.Analysis of cell cycles by flow cytometry,soft agar assay,cell adhesion assay,invasion assays,cytoskeleton staining(coomassie brilliant blue R250),morphological changes viewed by scanning electron microscope (SEM) and transmission electron microscope(TEM),metastasis assay through the lateral tail-vein injection,respectively.Metastasis assay in vivo by whole-body visualizing animal imaging system and H&E staining were used to evaluate the functional e alteration s of Tiam1-knockdown NPC cells in vivo.
4.Screening and identification of Tiam1-interacting proteins
To explore the Tiam1-mediated possible molecular mechanism of relating to invasion and metastasis in NPC cells,we firstly screened Tiam1-interacting protein in (http://www.genome.jp/kegg/) and(http://visant.bu.edu/),then we chose ankyrin1, rac1 and CD44 proteins which closely correlated to invasion and metastasis for co-localization analysis by double immunofluorescence staining and co-immunoprecipitation(CO-IP).
RESULTS
1.The expression of Tiam1 in nasopharyngeal carcinoma tissues and cell lines.
The results of IHC for Tiam1 were summarized as follow:The localization of Tiam1 protein is in cytoplasm and cytomembrane.The average scores for Tiam1 expression were significantly higher in NPC tissues than those in chronic nasopharyngitis tissues(t=7.224,P＜0.01);The level of Tiam1 protein in 6 tumor tissues from nude mice inoculated with metastatic human NPC cells was also higher. Comparison with the TNM stages of NPC patients,no difference was found in Tiam1 expression among NPC patients in different T stages(F=1.670,P=0.182),while the expressions differed significantly between the patients with lymph node metastasis and those without metastasis,and also between patients with organ metastasis and those without(t=9.005,P＜0.01;t=5.069,P＜0.01).The expression of Tiam1 in 6 NPC cell lines was detected in protein level by immunofluorescence staining and in mRNA level by RT-PCR respectively.The results were shown that higher-level Tiam1 was found in 5-8F cell line with higher metastasis property(0.560±0.020);In 6-10B cell line without metastasis ability,fewer Tiam1 was detected(0.110±0.010) and in others cell lines such as CNE1,CNE2,HONE1 and C666-1,moderate-expressed Tiam1 mRNA was detected.
2.Generation and functional verification of Tiam1-overexpressed CNE2 Cells.
We firstly generated CNE2 cells stably transfected with Tiam1/c1199 cDNA and empty vector(named as CNE2/Tiam1 or C/T,CNE2/Mock or T/M).RT-PCR and western-blot methods were used to analysis the expression of Tiam1 in four chosen Tiam1-transfected clones of CNE2.The highest level of Tiam1 expression was seen in the Tiam1 transfected clone3.We chose T3 clone as function experiments in vitro or vivo,and the results shew that Tiam1 overexpression promoted proliferation(MTT assay:F=323.158,P＜0.01;plate colony formation assay:t=25.065,P＜0.01;soft agar assay:F=318.960,P＜0.01),adhesive(F=86.689,P＜0.05) and invasive capabilities (F=323.158,P＜0.05) in CNE2/Tiam1 cells compared with CNE2 or CNE2/Mock in vitro.
To evaluate the role of Tiam1 overexpression on metastatic ability of NPC,we performed in vivo metastasis assay by tail-vein injection.On the fifteenth day after injection,one of the mice in CNE2/Tiam1 group was died from multiple abscesses. By the nineth week after injection,when nude mice had not died but some appeared to be moribund,all mice were sacrificed.Then their lungs,livers and other organs were removed and fixed in 10%formalin for H&E staining.We found that all 8 nude mice in CNE2/Tiam1 group emerged lung metastatic nodes,while in CNE2/Mock group,4 of 9 nude mice shew lung metastatic nodes under dissecting microscope.The average number of lung metastatic nodes in CNE2/Tiam1 group in each nude mice (7.75±2.315) was higher than that in CNE2/Mock group(3.78±4.549)(t'=2.305, P＜0.05).All the suspected metastatic nodes were confirmed by histopathological examination.In addition,scattered NPC cells infiltration in liver tissues was found in one of CNE2/Tiam1 group under microscope.No metastatic focus was found in the other organs.
3.Establishment and identification of lentiviral-mediated Tiam1-knockdown CNE2 cells
To require prolonged suppression of Tiaml protein in CNE2 cells,we constructed 4 shRNA(pGC-LV recombination vector) containing Tiam1 interfere sequence A,B,C and D.Real-time PCR analysis showed that the mRNA levels of Tiaml in four Tiam1-si cells were all suppressed,especially in Tiam1-si-B cells (0.124±0.055),next to si-C(0.156±0.046).Besides,we reinfected CNE2 cells with si-B,si-C lentivirus for further identifycation.On thirth days after transfection, fluorescence microscope photographs were firstly demonstrated the efficient transduction of CNE2 cells.The results in mRNA or protein levels by Real-time PCR and western-blot were consistent with above preliminary identification.We named Lenti-si-B,si-C infected CNE2 cells as KD1 and KD2 respectively.Furthermore,we chose KD1 cells as functional experiments in vitro or in vivo.
4.Functional alterations of lentiviral-mediated Tiam1-knockdown NPC cells.
To further detect the role of Tiam1 in NPC,we established Tiam1-knockdown CNE2 cells,and a series of assays were performed to evaluate the function alterations of Tiam1-knockdown cells in invasion and metastasis.Flow cytometry was used to detect the alteration of cell cycle in CNE2,CNE2/NC and CNE2/KD cells.The results showed that their percentages in S stage were 51.3%,52.7%and 21% respectively,which indicated that the ability of proliferation on CNE2 cell was declined after Tiam1 depletion.Soft agar assay was used to detect the abilitiy of colony formation and obtained declined the ability of proliferation of single Tiam1-knockdown CNE2 cells(F=645.863,P＜0.05).In order to further determine whether Tiam1 protein has a role in enhancing cell adhesion to fibronectin,we conducted cell adhesion assay and found that compared to CNE2 and CNE2/NC,the adhesive capability to fibronectin of CNE2/KD cells was decreased(F=55.388,P＜0.05). Results from the invasion assay showed the reduced ability of CNE2/KD cells to migrate through the matrigel membrane,and it demonstrated that suppression of Tiaml effectively reduced the invasive capability of CNE2 cells in vitro(F=181.727, P＜0.05).Besides,coomassie brilliant blue staining was used to compare cytoskeleton changes in the content and distribution of NPC cells.The results showed that the cytoskeleton in CON,NC cells were large,intensive and fascicularis arranged along with cell morphology;In contrast,in KD cells small and sporadic distribution of cytoskeleton were seen.Scanning electron microscopy(SEM) showed CON,NC cells closely attached to the slide,alive with in microvilli each other;Adversely, surface microvilli of KD cells were sparse,short,and observed disjunction phenomenon.Ultrastructural changes of cells were observed by transmission electron microscope(TEM).In KD cells,nuclear heterochromatin increased margination. Vesicular or lacunar endoplasmic reticulum and Golgi complex,and apoptotic bodies were viewed in comparision with CON and NC cells.
In addition,to evaluate the effects of Tiaml depletion on metastasis in vivo,we injected NC cells(n=9,groupl) and Tiam1-KD CNE2 cells(n=9,group2) into 6-8-week-old athymic mice through the lateral tail vein and detected.By the nineth week,all mice were executed and their metastatic nodules in the lung or other organs were firstly observed under a GFP imaging system,and then their lungs,livers and other organs were fixed in 10%formalin for further analysis of histopathology.We found that 5 of 9 mice in groups1 formed metastatic nodules in the lung of mice analysed(5/9,all together 36 foci).The average number in every mouse was(4.00±4.093).In contrast,only one lung micro metastatic focus was found in group2 under a GFP imaging system.The discrepancy between group1 and group2 was significant by statistical analysis(t'=2.841,P＜0.05);No other organ but lung was found metastatic nodules,indicating that Tiam1 depletion reverses cancer metastasis in vivo.
5.Co-localization analysis of Tiam1-interacting proteins
Double immunofluorescence staining was firstly used to confirm whether heterogenous Tiam1-Ankyrinl,Tiam1-Rac1 or Tiam1-CD44 were colocalized in CNE2 cells.Cells were simultaneously stained with rabbit-anti-Tiam1 antibodies and mouse-anti-Ankyrin1,Rac1,CD44 antibodies followed by TRITC and FITC conjugated antibodies;The results were observed under confocal microscopy: Tiaml protein presenting red fluorescence and Ankyrin protein showing green fluorescence were punctiformly colocalized in cytoplasm of CNE2;Tiam1 and Rac1 proteins were extensively colocalized in cytoplasm while Tiam1 and CD44 proteins were only filiformly colocalized in medial surface of cell membrane.In addition, co-immunoprecipitation(CO-IP) was also applied to further detect the co-localization of Tiam1-Ankyrin1,Tiam1-Rac1 or Tiam1-CD44.By following western-blot ananlysis,we only observed the co-localization of Tiam1-Ankyrin1 and Tiam1-Rac1, no co-localization was found from Tiam1-CD44 proteins.
CONCLUSION
1.Tiam1 gene is closely correlated to NPC progression,and may be a useful gene of NPC invasion and metastasis.
2.Tiam1 protein can directly interact with Ankyrinl protein,Rac1 protein in NPC cells,indicating that it is a key protein in signal pathways related to invasion and metastasis of NPC.
鼻咽癌(nasopharyngeal carcinoma,NPC)是我国南方及东南亚地区常见的一种恶性肿瘤,尤以广东省珠江三角洲一带多见,俗有“广东癌”之称。虽然近年来鼻咽癌的诊断和治疗水平已有很大提高,但鼻咽癌的5年生存率仍很低,究其原因,转移是导致鼻咽癌患者治疗效果和死亡的关键。随着分子生物学研究的进展,尤其是基因芯片技术的应用,目前已经探索出许多与鼻咽癌转移有关的分子标志物,如VEGF、CXCR4、CD44、Cadherin、MMP、nm-23、Wt-p53等,它们在鼻咽癌侵袭转移中均发挥重要作用。有关T淋巴瘤侵袭转移诱导因子1(T lymphoma invasion and metastasis inducing factor 1,Tiam1)基因在鼻咽癌侵袭转移中的功能及机制研究目前少有报道。
Tiam1基因是采用前病毒插入致突变的原理,在BW5147小鼠T淋巴瘤细胞高侵袭变异株中分离得到,与肿瘤生物学特性的关系已逐步成为目前的研究热点。研究表明,Tiam1在乳腺癌、肺癌、结直肠癌等多种肿瘤组织中表达,特别在低分化的和伴有局部浸润或远处转移的腺癌、鳞癌组织中呈强阳性表达。通过对来源于人和啮齿类肿瘤源性的40多个细胞系的检测发现,Tiam1在大多数肿瘤细胞中表达,但在来源于相同组织类型的不同肿瘤细胞系中Tiam1的表达依据其肿瘤侵袭转移能力的不同而异。另有研究认为,Tiam1基因过表达可促进结直肠癌细胞的侵袭转移作用,Tiam1基因沉默可降低结直肠癌细胞的侵袭转移能力。也有研究发现,Tiam1基因敲除的小鼠能够抵抗诱导的皮肤癌发生,即使产生肿瘤,其数量、生长速度及转移瘤发生率也远较野生型荷瘤鼠低。Tiam1诱导肿瘤细胞侵袭转移的分子机制与肿瘤细胞骨架、肿瘤细胞粘附分子、肿瘤细胞膜皱褶、片状伪足形成以及细胞外基质密不可分。还有研究表明,Ankyrin蛋白以其ADR(ankyrin repeat domain)区与Tiam1结合,通过Ankyrin-Tiam1-Rac1实现细胞骨架结构重塑,促进乳腺癌细胞的侵袭转移;癌基因CD44-V3可与Tiam1蛋白的PHn-CC-Ex功能域特异性结合,从而调节Tiam1-Rac信号,促进乳腺癌SP1细胞的侵袭转移。
浸润转移是鼻咽癌最重要的生物学特性之一,尽管Tiam1基因在多种肿瘤组织及肿瘤细胞系中均有表达且与肿瘤转移的关系已基本明确,但在人鼻咽癌中的表达、作用及机制目前国内少有文献报道,国外未见报道。因此,本研究结合临床资料,应用基因转染、基因沉默及体内外实验等研究方法,从人群和分子水平探讨Tiam1基因在鼻咽癌发生发展中的作用及其机制,为鼻咽癌转移的个体化基因治疗提供新靶标和新方法。
方法
1.Tiam1在鼻咽癌中的表达及其作用
应用免疫组化SP-9000法检测71例鼻咽未分化型非角化性癌、20例慢性鼻咽粘膜炎、6例人鼻咽癌细胞株裸鼠转移模型肿瘤组织中Tiam1蛋白的表达,同时运用免疫荧光和RT-PCR方法分别检测Tiam1在6种鼻咽癌细胞株中的表达。
2.Tiam1过表达对鼻咽癌细胞株侵袭转移特性的影响
根据Tiam1基因在6种人鼻咽癌细胞株中的表达情况,选择Tiam1中度表达、具有一定成瘤及转移能力的细胞株,利用阳离子脂质体LipofectamineTM2000法将Tiam1/C1199 cDNA质粒导入人鼻咽癌细胞,G418筛选抗性单克隆后扩大培养,用RT-PCR及Western blot方法鉴定转染后Tiam1基因在细胞中的表达情况并进行体内外功能验证实验。
3.Tiam1基因沉默对鼻咽癌细胞侵袭转移特性的影响
针对Tiam1(NM 003253)靶基因序列,利用公用网站、按照RNA干扰序列设计原则,设计A、B、C、D四个RNA干扰靶点序列,将含各干扰序列的双链DNA oligo粘性两端酶切(AgeI/EcoRI酶切)并连接到酶切后线性化的RNA干扰载体PGC-LV-GFP上。将连接好的产物转入制备好的细菌感受态细胞,对长出的克隆先进行菌落PCR鉴定,再进行测序比对,鉴定后的阳性克隆即为构建成功的目的基因RNA干扰慢病毒载体(即VshRNA)。将含各干扰序列的VshRNA质粒与pHelper1.0和pHelper2.0载体质粒共转染293T病毒包装细胞,收集富含慢病毒颗粒的细胞上清液,对其浓缩后得到高滴度的慢病毒浓缩液,在293T细胞中测定并标定病毒滴度。分别用含Tiam1各干扰序列的慢病毒感染人鼻咽癌细胞,同时设置阴性对照。用Real-time PCR方法初步筛选有效的干扰靶点,根据Real-time PCR初步筛选结果,选择干扰效果较好的两种病毒液再次感染CNE2细胞,待感染时间达到7天后收集细胞,进行荧光定量PCR和Westernblot鉴定。选取最有效的病毒液感染的鼻咽癌细胞用于后续的体内、体外功能验证实验。
4.Tiam1相关信号通路的初步探讨
利用基因通路网站(http://www.genome.jp/kegg/)、酵母双杂交网站(http://visant.bu.edu/)等得到若干与Tiam1相互作用的蛋白,从中选取与Tiam1侵袭转移特性密切相关的Ankyrin1、Rac1、CD44蛋白进行研究。运用免疫荧光双标记技术验证人鼻咽癌细胞中Tiam1-Ankyrin1、Tiam1-Rac1、Tiam1-CD44的表达并进行共定位分析;采用免疫共沉淀、免疫印迹技术验证Tiam1与CD44、Ankyrin1、Rac1蛋白的相互作用,探讨Tiam1在鼻咽癌侵袭转移中参与相关信号通路及其与CD44、Ankyrin1、Rac1蛋白的相互调控关系。
结果
1.鼻咽癌组织、鼻咽癌细胞株中Tiam1的表达
免疫组化结果表明,Tiam1蛋白在鼻咽癌组中的阳性表达得分为2.943±1.297,显著高于慢性炎症组0.700±0.923(t=7.224,P＜0.01);鼻咽癌组Tiam1蛋白表达得分与年龄无相关关系(r=0.020,P=0.871),其得分在性别之间、在T分期之间也没有统计学差异(t=1.702,P=0.093;F=1.670,P=0.182),而在有无淋巴结转移组间和有无远处器官转移组间差异具有统计学意义(t=9.005,P＜0.01;t=5.069,P＜0.01)。在具有转移特性的人源性鼻咽癌细胞株裸鼠模型肿瘤组织中Tiam1蛋白均呈高表达;运用免疫荧光和RT-PCR方法检测Tiam1在6种人鼻咽癌细胞株中的表达时发现,Tiam1在鼻咽癌高转移细胞株5-8F中表达(0.560±0.020)高于无转移特性的细胞株6-10B中的表达(0.110±0.010)(t=34.86,P＜0.01)。以上研究结果表明Tiam1与鼻咽癌的侵袭转移特性相关。
2.Tiam1稳定转染人鼻咽癌细胞株的建立及其侵袭转移特性的变化经RT-PCR及Western blot方法验证显示各转染克隆细胞中Tiam1的表达均高于未转染或空载体转染的鼻咽癌细胞,表明成功建立了Tiam1过表达的鼻咽癌细胞株。选取Tiam1表达最强的细胞克隆分别进行以下的体内外功能验证实验。
MTT实验结果证实,在细胞培养的第1天,转染了Tiam1的CNE2细胞的增殖能力与空载体转染组和未转染组比较没有统计学差异(F=0.740,P=0.498);随着细胞培养天数的增加,CNE2/Tiam1组细胞的增殖能力明显超过前两组(第2-7天各细胞组单因素方差分析的F值分别为3.289、15.519、3.616、58.265、28.983、57.459,P值分别为0.073、0.000、0.059、0.000、0.000、0.000),表明Tiam1过表达增强了鼻咽癌CNE2细胞的体外增殖能力。
平板克隆形成实验、软琼脂集落形成实验结果均显示,Tiam1过表达的CNE2细胞较未转染细胞其克隆生长能力增加,差异具有统计学意义(平板克隆形成实验:t=25.065,P＜0.01;软琼脂集落形成实验:F=318.960,P＜0.01)。
粘附实验结果表明,Tiam1基因过表达的CNE2细胞对FN的粘附能力高于未转染细胞和空载体转染细胞,差异具有统计学意义(F=86.689,P＜0.05)。
划痕试验检测Tiam1基因转染后对人鼻咽癌细胞株CNE2迁移能力的影响。方差分析结果是:Tiam1过表达增加了鼻咽癌细胞的迁移能力(F=356.753,P＜0.05)。
细胞侵袭实验证明,Tiam1转染细胞与空载体转染、未转染细胞相比较,其穿过基底膜的细胞数显著增多,差异具有统计学意义(F=323.158,P＜0.05)。
鼻咽癌裸鼠体内转移实验发现,在Tiam1转染组的8只裸鼠肺内均形成了明确的转移灶,在空载体转染组的9只裸鼠体内有4只形成肺内转移灶。Tiam1转染组每只裸鼠肺内所形成的转移灶个数(7.75±2.315)明显大于空载体转染组(3.78±4.549)(t′=2.305,P＜0.05)。其中在1只Tiam1过表达组的裸鼠肝组织内还可见散在的鼻咽癌细胞浸润。
3.Tiam1稳定沉默鼻咽癌细胞株的建立及其侵袭转移特性的改变
将含Tiam1干扰序列A、B、C、D的病毒液以及阴性对照的病毒液分别感染人鼻咽癌CNE2细胞,7天后收集细胞经过Real-time PCR的初步筛选得到,B序列干扰效果最好(0.124±0.055),C序列次之(0.156±0.045);A和D序列的干扰效果相对较低(A,0.230±0.043;D,0.255±0.116)。与未感染组和阴性对照组比较,差异具有统计学意义(F=69.732,P＜0.05)。为了确保结果的可靠性,又将含B、C干扰序列的慢病毒分别和混合感染人鼻咽癌CNE2细胞,培养7天后收集细胞再次进行Real-time PCR鉴定及Western blot鉴定,两种验证方法所得出的结果与初步筛选时所得出的结论相一致;并将感染B慢病毒的CNE2细胞命名为KD,感染阴性对照慢病毒的CNE2细胞命名NC,未感染的CNE2细胞命名CON,并进行以下的体内、体外功能验证实验。
采用流式细胞术分析三组细胞的细胞周期分布,结果发现Tiam1/KD组的CNE2细胞处于G1期的比例增高,处于S期和G2+M期细胞的比例则相应降低,与NC及CON组比较,差异具有统计学意义(F值分别为2021.007、1141.527、73.162,P＜0.05),表明Tiam1干扰后细胞的增殖能力下降。
软琼脂集落形成实验结果表明Tiam1/KD细胞其单个细胞的增殖能力较未干扰的鼻咽癌细胞降低,差异具有统计学意义(F=645.863,P＜0.05)。
异质粘附实验结果发现,Tiam1/KD细胞对FN的异质粘附能力下降,与未干扰的CNE2细胞比较,差异具有统计学意义(F=55.388,P＜0.05)。
采用细胞侵袭实验分析Tiam1干扰后细胞体外侵袭能力的变化,统计分析结果显示Tiam1/KD细胞穿过人工基底膜胶的侵袭细胞数量显著低于Tiam1未干扰的CNE2细胞(F=181.727,P＜0.05),表明Tiam1干扰后的CNE2细胞的体外侵袭能力降低。
运用考马斯亮蓝细胞骨架染色方法观察Tiam1干扰前后细胞骨架的含量及分布变化,结果表明Tiam1/KD组的CNE2细胞其细胞骨架密集,粗大,沿细胞形态走向排列,可见明显的细胞突起;而NC、CON组的未干扰细胞的细胞骨架呈稀疏、散在分布。
扫描电镜结果显示NC、CON组细胞与玻片贴附紧密,微绒毛丰富,相互间形成连接;而Tiam1干扰后的CNE2细胞则细胞表面微绒毛稀疏、短小,可见中断现象,进一步印证了细胞骨架染色的结果。
透射电镜观察Tiam1干扰前后细胞超微结构的变化,发现NC、CON组的CNE2细胞,其细胞核内染色质淡染、分布均匀,线粒体、内质网及高尔基复合体(Golgi complex)等细胞器结构完整正常;而Tiam1干扰后的KD细胞其核内异染色质增多,常染色质减少;胞浆内可见裂隙及囊状空泡,可见细胞凋亡小体形成。
采用尾静脉注射法建立了Tiam1沉默前后鼻咽癌细胞的裸鼠体内转移模型。2月后处死裸鼠并在整体荧光体视镜下观察发现在NC组的9只裸鼠中有5只出现了肺转移,共有36个肺转移结节,平均每只裸鼠肺转移结节数量为(4.00±4.093);在CNE2/KD组的9只裸鼠中仅有1只可观察到针尖大小的疑似肺内转移结节,差异具有统计学意义(t′=2.841,P＜0.05)。对各组裸鼠的其它器官组织切片观察显示两组裸鼠均未发现鼻咽癌细胞的转移。
4.鼻咽癌中Tiam1相互作用蛋白的验证
运用免疫荧光双标记技术验证人鼻咽癌CNE2细胞中Tiam1-Ankyrin1、Tiam1-Rac1、Tiam1-CD44的表达并进行共定位分析。结果显示Tiam1和Ankyrin1蛋白均在胞浆中分布,可见两种蛋白散点状共定位的棕黄色区域;Rac1蛋白在CNE2细胞中丰富表达,与Tiam1蛋白明显共定位于胞浆而呈现明亮的黄色;而在Tiam1-CD44实验组,Tiam1仅和CD44蛋白的胞内区域共定位,在细胞膜内侧面呈现较弱的线型黄色区域,在细胞膜的外侧面及胞膜区域则呈现CD44蛋白标记的绿色荧光。
免疫共沉淀实验进一步验证Tiam1与Ankyrin1、Rac1和CD44蛋白的相互作用,结果只检测到Tiam1与Ankyrin1、Rac1的共沉淀现象,没有检测到Tiam1与CD44蛋白共沉淀的发生。
结论
1.Tiam1基因与鼻咽癌进展密切相关,是促进鼻咽癌增殖、粘附、侵袭及转移的重要基因。
2.Tiam1、Ankyrin1和Rac1三种蛋白相互作用,可能共同参与并促进鼻咽癌侵袭转移的发生,Tiam1可能是鼻咽癌侵袭转移相关信号通路中的关键蛋白之本研究的创新之处
1.采用绿色荧光蛋白(GFP)标记的慢病毒载体和RNAi技术建立了稳定沉默Tiam1蛋白的鼻咽癌细胞系,为进一步研究Tiam 1蛋白在鼻咽癌侵袭转移中的功能和作用提供一个理想的实验平台。
2.利用免疫荧光双标记技术和免疫共沉淀方法对筛选出的Tiam1相互作用蛋白在鼻咽癌细胞中进行共定位分析,初步探讨了Tiam1在鼻咽癌侵袭转移中的相关信号通路。
3.较系统地探讨了Tiam1基因在鼻咽癌中的表达及其与鼻咽癌细胞增殖、粘附与侵袭转移的关系。
BACKGROUND & OBJECTIVE
Nasopharyngeal carcinoma(NPC) is a type of cancer common in Southern China and Southeast Asia,especially those of Cantonese origin.Although the levels of diagnosis and the treatment on NPC have being evidently improved in recent years, the survival rate of 5 years for patients with NPC is still very low,which the main cause of higher mortality is early lymph node and distant organ metastases.Recently, many molecular markers relating invasion and metastasis of NPC have been found such as VEGF,CXCR4,CD44,Cadherin,MMP,nm-23,Wt-p53,etc.They play an important role in NPC's invasion and metastasis.However,the function and mechanism of Tiam1(T lymphoma invasion and metastasis inducing factor 1)in NPC's invasion and metastasis are still unclear.
Tiam1 was identified in 1994 by proviral tagging in combination with in vitro selection for invasiveness from murine lymphoma cells.It had been reported that Tiam1 was extensively expressed in a variety of cancers,such as breast cancer,lung cancer,colorectal cancer,and so on,especially in adenocarcinoma and squamous cell cancer with local infiltration and/or distant metastasis.Moreover,examination on more than 40 rodent and human tumor cell lines demonstrated that Tiam1 expression in virtually all lines.Furthermore,the level of Tiam1 was positively correlated with metastatic potential of tumor cell lines.Another research found that Tiam1 overexpression promoted progression of colorectal cancer cell lines,while downregulating Tiam1 protein inhibited their bionomics such as the invasion and metastasis.Tiam1~(-/-) mice were resistant to the development of Ras-induced skin tumors.
Considerable work had demonstrated that the molecular mechanisms of Tiam1-induced invasion and metastasis were closely correlated to cystoskeleton rearrangement,cellular adhesion,cell cycle progression,gene transcription,formation of cell membrane ruffles,etc.Several recent studies had demonstrated that Tiaml binded to the cytoskeletal protein ankyrin,causing Tiam1-mediated Rac1 activation as well as an increase in breast tumor cell migration and invasion.Similarly,the cellular adhesion molecule CD44 binded to Tiam1 at the PHn-CC-Ex region in SP1 murine breast carcinoma cells.Furthermore,the binding of HA to CD44v3 causes an increase in Tiam1-mediated Rac activation and promoted cytoskeleton-mediated tumor cell migration.
Invasion and metastasis are the main bionomics and directly cause of invalid treatment or death of NPC.Up to now,few reports was found about function and mechanism of Tiam1 in NPC.In this study,we mainly focus on the functional alterations of Tiam1 in NPC tissues and cell lines by gene transfection,RNAi,etc.The aim is insighting into the role of Tiam1 in invasion and metastasis,and providing a molecular marker for NPC's treatment and prognosis.
METHODS
1.Expression of Tiam1 and it's relationship with biological behaviour of NPC.
Immunohistochemistry(SP-9000) was performed to examine the expressions of Tiam1 protein in 71 specimens of NPC tissue,20 specimens of chronic nasopharyngitis tissue,and 6 specimens of tumor tissues from nude mice inoculated with metastatic human NPC cells.The expression of Tiam1 in 6 NPC cell lines was detected in protein level by immunofluorescence staining and in mRNA level by RT-PCR respectively.
2.Establishment and functional verification of Tiam1-overexpressed NPC Cells.
We firstly established stably Tiam1-overexpressed CNE2 cells by transfecting Tiam1/c1199 cDNA with lipofectamin2000.The Tiaml-overexpressed CNE2 clones was screened with G418 and verified by RT-PCR and Western blot.The functional alterations of the resulting transfectants were performed by MTT assay,plate colony formation assay,soft agar assay,cell adhesion assay,cell wounding heal assay, invasion assays in vitro and metastasis assay in vivo through the lateral tail-vein injection.
3.Establishment of Tiam1-knockdown NPC cells and functional alterations in vitro and in vivo.
Lentiviral expression vectors containing enhanced green fluorescence protein (GFP) and Tiam1 small interfering RNA(Lenti-Tiam1si),or the control siRNA (Lenti-NC) gene were constructed.A human monocyte-derived cell line CNE2 was transfected with a different multiplicity of infection(MOI) of Lenti-Tiam1-si-A,B,C, D or Lenti-NC,and cultured to obtain stably-transfected CNE2-KD and CNE2-NC cells.The expression of Tiam1 mRNA was determined by real-time PCR,and Tiam1 protein was detected by western-blot.Analysis of cell cycles by flow cytometry,soft agar assay,cell adhesion assay,invasion assays,cytoskeleton staining(coomassie brilliant blue R250),morphological changes viewed by scanning electron microscope (SEM) and transmission electron microscope(TEM),metastasis assay through the lateral tail-vein injection,respectively.Metastasis assay in vivo by whole-body visualizing animal imaging system and H&E staining were used to evaluate the functional e alteration s of Tiam1-knockdown NPC cells in vivo.
4.Screening and identification of Tiam1-interacting proteins
To explore the Tiam1-mediated possible molecular mechanism of relating to invasion and metastasis in NPC cells,we firstly screened Tiam1-interacting protein in (http://www.genome.jp/kegg/) and(http://visant.bu.edu/),then we chose ankyrin1, rac1 and CD44 proteins which closely correlated to invasion and metastasis for co-localization analysis by double immunofluorescence staining and co-immunoprecipitation(CO-IP).
RESULTS
1.The expression of Tiam1 in nasopharyngeal carcinoma tissues and cell lines.
The results of IHC for Tiam1 were summarized as follow:The localization of Tiam1 protein is in cytoplasm and cytomembrane.The average scores for Tiam1 expression were significantly higher in NPC tissues than those in chronic nasopharyngitis tissues(t=7.224,P＜0.01);The level of Tiam1 protein in 6 tumor tissues from nude mice inoculated with metastatic human NPC cells was also higher. Comparison with the TNM stages of NPC patients,no difference was found in Tiam1 expression among NPC patients in different T stages(F=1.670,P=0.182),while the expressions differed significantly between the patients with lymph node metastasis and those without metastasis,and also between patients with organ metastasis and those without(t=9.005,P＜0.01;t=5.069,P＜0.01).The expression of Tiam1 in 6 NPC cell lines was detected in protein level by immunofluorescence staining and in mRNA level by RT-PCR respectively.The results were shown that higher-level Tiam1 was found in 5-8F cell line with higher metastasis property(0.560±0.020);In 6-10B cell line without metastasis ability,fewer Tiam1 was detected(0.110±0.010) and in others cell lines such as CNE1,CNE2,HONE1 and C666-1,moderate-expressed Tiam1 mRNA was detected.
2.Generation and functional verification of Tiam1-overexpressed CNE2 Cells.
We firstly generated CNE2 cells stably transfected with Tiam1/c1199 cDNA and empty vector(named as CNE2/Tiam1 or C/T,CNE2/Mock or T/M).RT-PCR and western-blot methods were used to analysis the expression of Tiam1 in four chosen Tiam1-transfected clones of CNE2.The highest level of Tiam1 expression was seen in the Tiam1 transfected clone3.We chose T3 clone as function experiments in vitro or vivo,and the results shew that Tiam1 overexpression promoted proliferation(MTT assay:F=323.158,P＜0.01;plate colony formation assay:t=25.065,P＜0.01;soft agar assay:F=318.960,P＜0.01),adhesive(F=86.689,P＜0.05) and invasive capabilities (F=323.158,P＜0.05) in CNE2/Tiam1 cells compared with CNE2 or CNE2/Mock in vitro.
To evaluate the role of Tiam1 overexpression on metastatic ability of NPC,we performed in vivo metastasis assay by tail-vein injection.On the fifteenth day after injection,one of the mice in CNE2/Tiam1 group was died from multiple abscesses. By the nineth week after injection,when nude mice had not died but some appeared to be moribund,all mice were sacrificed.Then their lungs,livers and other organs were removed and fixed in 10%formalin for H&E staining.We found that all 8 nude mice in CNE2/Tiam1 group emerged lung metastatic nodes,while in CNE2/Mock group,4 of 9 nude mice shew lung metastatic nodes under dissecting microscope.The average number of lung metastatic nodes in CNE2/Tiam1 group in each nude mice (7.75±2.315) was higher than that in CNE2/Mock group(3.78±4.549)(t'=2.305, P＜0.05).All the suspected metastatic nodes were confirmed by histopathological examination.In addition,scattered NPC cells infiltration in liver tissues was found in one of CNE2/Tiam1 group under microscope.No metastatic focus was found in the other organs.
3.Establishment and identification of lentiviral-mediated Tiam1-knockdown CNE2 cells
To require prolonged suppression of Tiaml protein in CNE2 cells,we constructed 4 shRNA(pGC-LV recombination vector) containing Tiam1 interfere sequence A,B,C and D.Real-time PCR analysis showed that the mRNA levels of Tiaml in four Tiam1-si cells were all suppressed,especially in Tiam1-si-B cells (0.124±0.055),next to si-C(0.156±0.046).Besides,we reinfected CNE2 cells with si-B,si-C lentivirus for further identifycation.On thirth days after transfection, fluorescence microscope photographs were firstly demonstrated the efficient transduction of CNE2 cells.The results in mRNA or protein levels by Real-time PCR and western-blot were consistent with above preliminary identification.We named Lenti-si-B,si-C infected CNE2 cells as KD1 and KD2 respectively.Furthermore,we chose KD1 cells as functional experiments in vitro or in vivo.
4.Functional alterations of lentiviral-mediated Tiam1-knockdown NPC cells.
To further detect the role of Tiam1 in NPC,we established Tiam1-knockdown CNE2 cells,and a series of assays were performed to evaluate the function alterations of Tiam1-knockdown cells in invasion and metastasis.Flow cytometry was used to detect the alteration of cell cycle in CNE2,CNE2/NC and CNE2/KD cells.The results showed that their percentages in S stage were 51.3%,52.7%and 21% respectively,which indicated that the ability of proliferation on CNE2 cell was declined after Tiam1 depletion.Soft agar assay was used to detect the abilitiy of colony formation and obtained declined the ability of proliferation of single Tiam1-knockdown CNE2 cells(F=645.863,P＜0.05).In order to further determine whether Tiam1 protein has a role in enhancing cell adhesion to fibronectin,we conducted cell adhesion assay and found that compared to CNE2 and CNE2/NC,the adhesive capability to fibronectin of CNE2/KD cells was decreased(F=55.388,P＜0.05). Results from the invasion assay showed the reduced ability of CNE2/KD cells to migrate through the matrigel membrane,and it demonstrated that suppression of Tiaml effectively reduced the invasive capability of CNE2 cells in vitro(F=181.727, P＜0.05).Besides,coomassie brilliant blue staining was used to compare cytoskeleton changes in the content and distribution of NPC cells.The results showed that the cytoskeleton in CON,NC cells were large,intensive and fascicularis arranged along with cell morphology;In contrast,in KD cells small and sporadic distribution of cytoskeleton were seen.Scanning electron microscopy(SEM) showed CON,NC cells closely attached to the slide,alive with in microvilli each other;Adversely, surface microvilli of KD cells were sparse,short,and observed disjunction phenomenon.Ultrastructural changes of cells were observed by transmission electron microscope(TEM).In KD cells,nuclear heterochromatin increased margination. Vesicular or lacunar endoplasmic reticulum and Golgi complex,and apoptotic bodies were viewed in comparision with CON and NC cells.
In addition,to evaluate the effects of Tiaml depletion on metastasis in vivo,we injected NC cells(n=9,groupl) and Tiam1-KD CNE2 cells(n=9,group2) into 6-8-week-old athymic mice through the lateral tail vein and detected.By the nineth week,all mice were executed and their metastatic nodules in the lung or other organs were firstly observed under a GFP imaging system,and then their lungs,livers and other organs were fixed in 10%formalin for further analysis of histopathology.We found that 5 of 9 mice in groups1 formed metastatic nodules in the lung of mice analysed(5/9,all together 36 foci).The average number in every mouse was(4.00±4.093).In contrast,only one lung micro metastatic focus was found in group2 under a GFP imaging system.The discrepancy between group1 and group2 was significant by statistical analysis(t'=2.841,P＜0.05);No other organ but lung was found metastatic nodules,indicating that Tiam1 depletion reverses cancer metastasis in vivo.
5.Co-localization analysis of Tiam1-interacting proteins
Double immunofluorescence staining was firstly used to confirm whether heterogenous Tiam1-Ankyrinl,Tiam1-Rac1 or Tiam1-CD44 were colocalized in CNE2 cells.Cells were simultaneously stained with rabbit-anti-Tiam1 antibodies and mouse-anti-Ankyrin1,Rac1,CD44 antibodies followed by TRITC and FITC conjugated antibodies;The results were observed under confocal microscopy: Tiaml protein presenting red fluorescence and Ankyrin protein showing green fluorescence were punctiformly colocalized in cytoplasm of CNE2;Tiam1 and Rac1 proteins were extensively colocalized in cytoplasm while Tiam1 and CD44 proteins were only filiformly colocalized in medial surface of cell membrane.In addition, co-immunoprecipitation(CO-IP) was also applied to further detect the co-localization of Tiam1-Ankyrin1,Tiam1-Rac1 or Tiam1-CD44.By following western-blot ananlysis,we only observed the co-localization of Tiam1-Ankyrin1 and Tiam1-Rac1, no co-localization was found from Tiam1-CD44 proteins.
CONCLUSION
1.Tiam1 gene is closely correlated to NPC progression,and may be a useful gene of NPC invasion and metastasis.
2.Tiam1 protein can directly interact with Ankyrinl protein,Rac1 protein in NPC cells,indicating that it is a key protein in signal pathways related to invasion and metastasis of NPC.
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