结直肠癌转移相关基因Tiam1功能的初步研究
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摘要
研究背景和目的
结直肠癌是常见的恶性肿瘤之一，近十年来，结直肠癌在我国的发病率呈逐年上升的趋势。转移是影响患者治疗效果和死亡的主要原因，探讨与结直肠癌转移相关的因素，寻找预防和治疗的有效途径，是当代肿瘤学研究的一项迫切任务。
在我们的前期研究中，利用自行研制的含有447个已知肿瘤转移相关基因cDNA芯片，筛选出51个结直肠癌转移相关基因。应用文献轮廓挖掘微阵列表达数据的生物信息学方法，筛选出4个与结直肠癌转移密切相关的新基因，并对其中一个新的结直肠癌转移相关基因—T淋巴瘤侵袭转移诱导因子1(T lymphoma invasion and metastasis，Tiaml)，在RNA水平进行了初步验证。
Tiaml是鸟苷酸转换因子(guanine nucleotide exchange factors，GEFs)家族成员，GEFs的主要功能是调节Rho GTPase的活性。Rho GTPase家族包括Rho、Rac及Cdc42，是Ras超家族的成员。Tiaml体内外均可以特异性地激活Rho GTPase家族中的Racl。新近的研究发现Tiaml不但激活Rac，更重要的是Tiaml直接与胞浆和胞膜上的蛋白质相互作用，将它们耦合到Tiaml-Rac信号通路上，从而影响Rac信号通路的特异性。
Tiaml基因最初在小鼠T淋巴瘤细胞高侵袭变异株中分离鉴定。随后，在乳腺癌、肺癌及Ras诱导的皮肤癌等肿瘤证实Tiaml具有明显的促进肿瘤进展和转移的作用。在Tiaml敲除的动物模型中，小鼠皮肤癌发生率明显减低并且肿瘤进展缓慢，Malliri等认为Tiaml在Ras诱导皮肤癌发生的启动和进展阶段发挥关键作用，这一效应与Tiam1表达量有明显的关系。上述的研究表明，Tiam1是多种肿瘤的促癌基因和促进转移基因。
本研究重点探讨Tiam1基因对结直肠癌增殖、侵袭及转移的影响和作用机制，阐明Tiam1在结直肠癌转移中的主要生物学功能，为结直肠癌转移的早期诊断和治疗奠定理论基础。
方法
1．结直肠癌组织中Tiam1的表达及临床意义
应用免疫组化S-P法，检测157例结直肠癌和配对的30例正常结直肠黏膜、35例结直肠癌区域淋巴结转移癌以及32例结直肠腺瘤组织中Tiam1蛋白的表达。
2．Tiam1过表达对结直肠癌细胞生物学特性的影响
用RT-PCR方法检测Tiam1基因在9种结直肠癌细胞株中的表达，筛选Tiam1不表达的细胞株；将Tiam1／C1199HA cDNA导入内源性不表达Tiam1基因的人结直肠癌HT29细胞株中，G418筛选抗性单克隆，RT-PCR、免疫组化及Western blot鉴定转染后Tiam1基因在细胞中的表达；观察Tiam1基因转染前后，细胞增殖、体外侵袭、成瘤能力及体内转移能力的变化。
3．建立整体可视化结肠癌原位及转移动物模型
利用阳离子脂质体Lipofectamine~(TM)2000将pEGFP-N1质粒导入人结直肠癌SW480细胞，获得稳定表达绿色荧光蛋白(enhanced green fluorescent protein，EGFP)的细胞株SW480／EGFP~+；通过结肠癌原位手术移植的方法建立结肠癌原位及转移模型，利用肿瘤细胞表达EGFP的特点，借助整体荧光体视镜结合IPP5.0软件采集、分析EGFP发出的荧光信号，在动物活体实时观察结肠癌局部成瘤及转移情况；利用MTT法检测和分析EGFP对细胞增殖能力的影响；应用病理学方法对结肠癌原位及转移动物模型进行评价和鉴定。
4．Tiam1表达沉默对结直肠癌细胞生物学特性的影响
设计Tiam1干扰片段，构建含U6启动子的慢病毒载体，将慢病毒载体质粒与辅助质粒共转染293FT病毒包装细胞，收集病毒上清，进行病毒滴度测定，用慢病毒感染结直肠癌细胞，用RT-PCR和Western blot鉴定有效的干扰载体。
用含Tiam1特异性干扰片段的慢病毒感染SW480／EGFP~+细胞，用含空载体的病毒做对照。用杀稻瘟菌素筛选抗性克隆，挑取抗性单克隆，荧光定量PCR和Western blot鉴定干扰后Tiam1基因在细胞中的表达；利用MTT法、平板克隆形成实验检测Tiam1对细胞体外增殖的影响、体外侵袭实验检测Tiam1对结直肠癌侵袭能力的影响、在裸鼠体内观察Tiam1对结直肠癌细胞致瘤能力的影响及利用可视化结直肠癌原位动物模型观察Tiam1对结直肠癌细胞体内转移能力的影响。
5．利用基因芯片和比较蛋白质组学技术分析Tiam1在结直肠癌增殖、侵袭和转移中的作用
利用Affymetrix公司的寡核苷酸芯片(47，400个转录本，包含38，500个已知基因)检测Tiam1基因表达沉默后差异基因表达谱。应用Gene Ontology、MILANO、Panther、Genomatix等软件进行生物信息学分析。
利用蛋白质组学技术分离HT29／Tiam1和HT29／mock细胞、SW480／EGFP~+／Tiam1~-和SW480／EGFP~+／mock细胞的总蛋白，建立重复性和分辨率较好的细胞双向凝胶电泳图谱，应用基质辅助激光解吸电离飞行时间质谱鉴定差异表达的蛋白质点。利用软件过滤基线峰、识别信号峰，利用Matrixscience公司的在线软件Mascot Distiller进行搜索分析。
利用荧光定量PCR、Western blot方法对所获得的差异基因和／或差异蛋白质进行鉴定。
结果
1．结直肠癌组织中Tiam1蛋白的表达
免疫组化结果显示Tiam1在正常结直肠黏膜、腺瘤、癌和淋巴结转移癌四种类型的组织中表达明显不同，差异具有显著性(x~2=23.561，P＜0.01)，两两比较发现，腺瘤Tiam1表达比正常结直肠黏膜组织强(Z=-2.423，P＜0.05)，淋巴结转移癌组织Tiam1表达比结直肠癌组织强(Z=-2.051，P＜0.05)，而结直肠癌组织与腺瘤组织Tiam1表达差异无显著性(Z=-0.938，P＞0.05)。在结直肠癌组织中，伴发区域淋巴结转移的癌组织比未发生转移的癌组织Tiam1表达明显增强，差异具有显著性(Z=-3.176，P＜0.01)。研究结果提示，Tiam1蛋白表达与结直肠癌转移高度相关，即Tiam1表达弱或阴性者转移的发生率低，表达强者其转移发生率高。
2．Tiam1质粒的转染及建立稳定表达Tiam1的结直肠癌细胞株
应用RT-PCR检测9种结直肠癌细胞株Tiam1基因的表达，发现HT29细胞内源性不表达Tiam1基因。将Tiam1质粒导入HT29细胞中，挑取G418抗性单克隆，RT-PCR及Western blot确定Tiam1在转染克隆1中表达最强(命名为HT29／Tiam1)，Tiam1在空载体对照中不表达(命名为HT29／mock)。
3．Tiam1过表达对结直肠癌细胞生物学特性的影响
通过MTT法，我们检测了Tiam1对结直肠癌细胞体外增殖能力的影响，与HT29／mock细胞相比，HT29／Tiam1细胞的增殖能力明显增强(F=239.197，P＜0.01)。此外，接种HT29／Tiam1细胞的裸鼠皮下肿瘤生长明显加快(F=48.410，P＜0.01)，肿瘤体积自皮下注射后的第七天起出现显著性差异，接种HT29／Tiam1细胞的裸鼠皮下肿瘤第20天的平均体积是HT29／mock细胞组的2.5倍。进行结直肠癌细胞外科原位移植6周后，接种HT29／Tiam1细胞的裸鼠结肠原位肿瘤重量明显大于接种HT29／mock细胞的裸鼠(t=-14.916，P＜0.01)。
体外侵袭实验证明，肿瘤细胞侵袭实验24h后，HT29／mock和HT29／Tiam1细胞均穿透基底膜向下侵袭，在聚碳酸酯膜上可见变形的肿瘤细胞，HT29／Tiam1细胞侵袭能力是HT29／mock细胞的1.9倍，说明Tiam1基因的表达增强了结直肠癌细胞的侵袭能力。
采用外科原位移植的方法比较HT29／mock和HT29／Tiam1细胞的体内转移能力。在HT29／Tiam1细胞组，100％(7／7)裸鼠发生了腹腔转移、57.1％(4／7)发生了肝转移、其中有一只裸鼠发生了广泛转移，包括脾、肺及淋巴结等转移。在HT29／mock细胞组，只有29％(2／7)裸鼠发生了腹腔转移，没有一只裸鼠发生远处转移。
4．整体可视化结肠癌原位及转移动物模型观察
本研究建立的结直肠癌细胞株SW480／EGFP~+稳定、高效的表达绿色荧光蛋白，且增殖能力与SW480相比，没有显著性差异(F=1.171，P＞0.05)；采用外科手术原位移植的方法建立结肠癌可视化原位动物模型，成功率达到100％。移植2周后，结肠原位成瘤，未发现有转移情况，手术6周后，75％的裸鼠发生了腹腔转移，50％发生了肝转移。通过病理学方法，验证了可视化原位及转移动物模型的可靠性。
5．通过RNAi技术沉默结直肠癌细胞株Tiam1表达，建立Tiam1基因稳定沉默的结直肠癌细胞株
构建了四个干扰Tiam1基因表达的慢病毒干扰载体，将慢病毒质粒和辅助质粒共转染293FT病毒包装细胞，收获病毒上清，测定病毒滴度为7×10~5Transducing Unit(TU)／ml。利用慢病毒感染SW480／EGFP~+细胞，荧光定量PCR和Western blot结果发现含片段B的慢病毒干扰载体的干扰效率最高(73％)。利用含干扰片段B的慢病毒和空载体病毒感染SW480／EGFP~+细胞，挑取杀稻瘟菌素抗性单克隆，荧光定量PCR和Western blot结果发现Tiam1在干扰克隆2中表达降低最明显(命名为SW480／EGFP~+／Tiam1~-)，干扰效率达88％。
6．Tiam1基因表达沉默后对结直肠癌细胞生物学特性的影响
MTT法观察Tiam1基因表达沉默后体外细胞的增殖情况，与SW480／EGFP~+／mock和SW480／EGFP~+细胞相比，SW480／EGFP~+／Tiam1~-细胞的增殖速度明显减慢，并且呈时间依赖关系(F=3.361，P＜0.01)。平板克隆形成实验显示SW480／EGFP~+／Tiam1~-细胞的活力显著下降，差异有统计学意义(F=7.244，P＜0.05)。这些结果均说明Tiam1表达水平减低后，肿瘤细胞体外生长被显著抑制。
体外侵袭小室检测Tiam1基因表达沉默后细胞侵袭能力的改变，结果显示，与SW480／EGFP~+／mock和SW480／EGFP~+细胞相比，SW480／EGFP~+／Tiam1~-细胞的侵袭能力明显降低，差异具有显著性(F=45.645，P＜0.01)，说明Tiam1表达沉默明显抑制了结直肠癌细胞的体外侵袭能力。
通过结直肠癌可视化动物模型，我们观察了Tiam1基因表达沉默后对结直肠癌增殖和转移能力的影响。将结直肠癌细胞接种于裸鼠的皮下，通过30天连续的观察，我们发现Tiam1基因表达沉默后，显著性地抑制了结直肠癌细胞的体内增殖能力(F=2.256，P＜0.05)，增殖差异从第15天开始，到第30天，差异达到1.3倍。
将Tiam1表达沉默的细胞接种于裸鼠结肠原位后，细胞的转移能力受到了限制。在SW480／EGFP~+／mock组，75％(6／8)裸鼠发生了腹腔转移，37.5％(3／8)裸鼠出现肝转移，12.5％(1／8)形成肺转移，而在SW480／EGFP~+／Tiam1~-组只有37.5％(3／8)形成腹腔转移，未发现裸鼠形成远处转移。
7．利用基因芯片技术探讨Tiam1在结直肠癌增殖、侵袭、转移中的作用
利用Affymetrix公司的HG-U133 plus 2寡核苷酸芯片对SW480／EGFP~+／Tiam1~-和SW480／EGFP~+／mock细胞的基因表达谱进行分析，获得Tiam1相关基因1026个，包括与Tiam1作用协同的基因647个，作用拮抗的基因379个。对获得的基因进行功能分类发现，与Tiam1基因协同作用的基因主要涉及了细胞周期、细胞周期调控、DNA复制、核苷酸代谢、调节细胞生理过程等，与Tiam1基因拮抗作用的基因主要涉及了生物过程负调控、抑制酶活性、细胞过程的负调控基因等。对获得的基因进行信号通路分类发现，参与Tiam1调节作用的基因主要涉及了Wnt通路、p53通路、凋亡信号通路、整合素信号通路、P13K信号通路、Ras信号通路等。
8．利用比较蛋白质组学技术分析Tiam1在结直肠癌增殖、侵袭、转移中的功能
比较蛋白质组学是当前蛋白质组学研究的主要策略，通过比较细胞在基因转染前后／沉默前后蛋白质表达差异，发现与目的基因作用相关的蛋白质。在本研究中，与HT29／mock细胞相比，HT29／Tiam1细胞表达的蛋白质在数量和表达水平上发生了一定变化，而这些发生改变的蛋白质与Tiam1的促进转移作用相关；SW480／EGFP~+／Tiam1~-细胞为通过RNAi技术使Tiam1表达沉默的细胞，其表达的蛋白质与SW480／EGFP~+／mock细胞相比，在数量和表达水平上亦发生了一定的变化，而这些发生改变的蛋白质亦与Tiam1的促进转移作用相关。因此，利用比较蛋白质组学的方法，分析这些细胞蛋白质表达的变化，可发现与Tiam1作用相关的蛋白质，从而了解其发挥作用的分子机制。在本研究中，利用蛋白质组学技术分离细胞的总蛋白，结合图像分析和基质辅助激光解析电离飞行时间质谱对部分差异表达的蛋白质点进行鉴定，共鉴定了20个差异表达的蛋白质点。我们对鉴定的蛋白质的功能进行分类，主要为与细胞代谢、细胞增殖、细胞骨架结构相关的蛋白，以及分子伴侣和未知功能的蛋白。这些差异蛋白质中有一些同肿瘤的增殖、转移密切相关。
9．Tiam1差异基因和／或蛋白质筛选过程中的转录组和蛋白质组的对接
在本研究中，我们利用基因芯片和比较蛋白质组学技术筛选与Tiam1作用的基因和／或蛋白质，转录组和蛋白质组对接的基因／蛋白质有3个，2个是与Tiam1基因功能协同的高迁移率族蛋白B1(high motility group box 1，HMGB1)和磷酸甘油酸变位酶(phosphoglycerate mutase，PGAM1)，他们随着Tiam1基因的表达上调而上调，表达沉默而下调；另一个是与Tiam1基因功能拮抗的膜联蛋白4(annexin A4，ANXA4)，它随着Tiam1基因的表达上调而下调，表达沉默而上调。
结论
1．Tiam1基因是结直肠癌增殖、侵袭、转移的促进基因；
2．Tiam1可能参与多条信号通路来介导它在结直肠癌中的作用，在相互作用的基因中HMGB1、PGAM1和ANXA4与Tiam1关系密切，他们是Tiam1信号通路中的新基因。本研究的创新之处
1．建立了整体可视化结直肠癌原位及转移动物模型，为结直肠癌的实验研究提供了理想的实验工具；
2．利用慢病毒载体和RNAi技术建立了稳定的Tiam1基因表达沉默的结直肠癌细胞株及Tiam1基因表达沉默的结直肠癌原位动物模型，为结直肠癌转移机制研究提供实验手段；
3．将芯片技术、比较蛋白质组学技术相结合，实现了转录组和蛋白质组的对接；
4．将RNAi技术、基因芯片技术、蛋白质组学技术相结合，用于Tiam1基因的功能研究，为阐明Tiam1在肿瘤转移中的作用提供新的证据，并综合分析Tiam1的信号转导通路，发现3个参与Tiam1信号通路新的成员。
BACKGROUND & OBJECTIVEColorectal cancer (CRC) is the second worldwide leading cause of cancer death in the world. The incidence rate of CRC in china is increasing fast during the past decades. Metastasis is one of the basic characteristic of malignant tumors and is the main cause which affects the therapeutic efficacy and leads to the death of cancer patients. It is an urgent task to work out the metastasis-associated factors and find out the preventive and therapeutic methods.In our previous studies, we prepared a cDNA microarray consisting of 447 tumor metastasis-associated candidate genes to screen metastasis-associated genes and obtained 51 candidate genes closely related to metastasis of colorectal cancer, four genes of which were selected as candidate metastatic genes of colorectal cancer by literature mining. Tiam1, one of the four candidate metastatic genes, was validated in the RNA level.Tiam1 is one of guanine nucleotide exchange factors (GEFs), which activate GTPases by promoting the exchange of their inactive GDP-bound forms to their active GTP-bound forms. Whereas Tiam1 displays GEF activity towards all three Rho-like GTPases Rac1, Cdc42 and RhoA in vitro, Tiam1 specifically activates Rac in vivo. Recent evidence suggests that Tiam1 could influence Rac GTPases signaling specificity in addition to promoting their activation. Tiam1 has been implicated to directly bind to many different cytoplasmic and membrane-associated proteins, which couples Tiam1-Rac activity to specific signaling pathways.
Tiam1 was originally identified as the invasion- and metastasis-inducing gene by proviral tagging in combination with in vitro selection for invasiveness in T lymphoma cells. The role of Tiam 1 in cellular migration, invasion and metastasis may not be limited to T lymphoma. It was reported to be important in promoting the tumor progression in a variety of cancers such as breast cancer, lung cancer and Ras-induced skin tumors. The possible role of Tiam1 in the metastasis of colorectal cancer is unclear.
In this study, we aim to clarify the possible role of Tiam1 gene in the proliferation, invasion and metastasis of CRC. It will be helpful to understand the molecular basis of CRC, and establish Tiam1 as a new target for early metastatic diagnostic markers and novel therapeutic strategies.
METHODS
1. Expression of Tiam1 in colorectal carcinomas and its clinical significance
The expression of Tiam1 was examined in specimens of 157 CRC, 30 paired nomal colorectal mucosae, 35 regional lymphatic metastases and 32 adenoma by immunohistochemical (S-P) method.
2. Effect of Tiam1 overexpression on the biological behaviors of human CRC
The expressions of Tiam1 gene in nine colorectal carcinoma cell lines were detected by reverse transcriptase polymerase chain reaction (RT-PCR). Tiam1/C1199HA cDNA was transfected into HT29, which had no endogenous Tiam1 expression. The Tiam1 expression in transfectant was determined by RT-PCR, immunohistochemistry and Western blot. The biological behaviors of tranfectant were investigated by MTT assay, in vitro invasion assay, tumorigenesis, and in vivo metastasis assay by surgical orthotopic transplantation (SOI).
3. Building the visualizing orthotopic animal model and observe real-time tumor growing and metastasis in colon cancer
The pEGFP-N1 plasmid was transfected into human colon carcinoma cell line SW480 to establish SW480/EGFP~+ cell line. MTT assay was engineered to detect the effect of EGFP on cell proliferation. The SW480/EGFP~+ cells were surgically orthotopically implanted as tissue fragments in the body of the colon of nude mice. Emitted fluorescence was collected and imaged through LT-9MACIMSYSPULS system and images were analyzed with IPP5.0 software. Whole-body optical images visualized real-time primary tumor growth and formation of metastatic lesions. The evaluation of orthotopic animal model was carried out with traditionally pathological methods.
4. Lentivirus-mediated silencing of Tiam1 gene in human CRC cell line
We transfected human CRC cell lines, SW480/EGFP~+ with Tiam1 specific RNAi lentiviral vectors as well as controls. Tiam1 silenced cells were screened by blasticidin. Quantitative RT-PCR and Western blot analysis were used to detect the expressions of Tiam1 mRNA and protein, respectively. MTT assay, plate colony formation assay, in vitro invasive assay, tumorigenesis, metastasis assay in vivo by whole-body visualizing orthotopic animal model of CRC were used to assess the functional effects of Tiam1 silencing on tumor cell proliferation, invasion and metastasis.
5. Exploring the possible molecular mechanism underlying Tiara1-mediated multiple functions in CRC using gene microarray and comparative proteomics method
Oligonucleotide microarray analysis was used to detect differences in gene expression between Tiam1-silencing SW480/EGFP~+ cells and the control.
Comparative two-dimensional gel electrophoresis (2-DE) technology was performed to separate the proteins between HT29/Tiam1 and HT29/mock cells and between SW480/EGFP~+/Tiam1~- and SW480/EGFP~+/mock cells respectively. Differentially expressed proteins were identified by Matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF-MS).
RESULTS
The main results and findings are as follows:
1. Expression of Tiam1 in CRC
Tiam1 was expressed heterogeneously among the normal colorectal mucosae, adenoma, carcinomas and lymphatic metastasis of colorectal carcinoma (x~2=23.561, P＜0.01). Tiam1 was higher expressed in adenoma compared with normal colorectal epithelium (Z=-2.423, P＜0.05). Tiam1 expression was significantly stronger in lymphatic metastasis in comparison with colorectal carcinoma (Z=-2.051, P＜0.05). Furthermore, the Tiam1 expression in colorectal carcinoma with lymph node metastasis is higher than that in colorectal carcinoma without lymph node metastasis (Z=-3.176, P＜0.01).
2. Transfection of C1199Tiam1 cDNA and selection of stable clones
To address the function of Tiam1 in CRC, we established stably transfected HT29 cells that overexpress Tiam1. As revealed by RT-PCR analysis, a higher level of Tiam1 expression was seen in the Tiam1 transfected clonel but not in the vector-only transfected clonel. The western blotting analysis also showed that Tiam1 expression in Tiam1 transfected clonel (designated HT29/Tiam1) was positive and vector-only transfected clonel (designated HT29/mock) was negative.
3. Effect of Tiam1 overexpression on the biological behaviors of human CRC
HT29/Tiam1 cells showed a significantly enhanced proliferation compared with the HT29/mock cells as determined by in vitro MTT assay. Next, the effect of Tiam1 on in vivo tumor growth was assessed by subcutaneous injection of HY29/Tiam1 and HT29/mock cells for twenty days. Compared with HT29/mock cells, the expression of Tiam1 led to a pronounced increase in HT29/Tiam1 cell growth starting from day 7, up to 2.5 fold increase in mice of tumor volume at day 20 after cell injection. Primary tumor growth after in vivo orthotopic implantation was also tested. In this assay, mice were sacrificed six weeks later, and tumors from surgical orthotopic implantation excised and weighed in HT29/Tiam1 group and HT29/mock group respectively. The result showed that Tiam1 expression led to a significant enhancement in orthotopic tumor growth.
Invasion through the extracellular matrix (ECM) is an important step in tumor metastasis. The ECMatrix served as a reconstituted basement membrane matrix of proteins. The number of cells migrating the ECMatrix was counted and the result showed that the HT29/Tiam1 cells posses a remarkable increase in invasiveness as compared with HT29/mock cells (P＜0.01). A 1.9-fold increase in the number of HT29/Tiam1 cells with an invasive ability was observed. These data indicated that Tiam1 expression in colorectal cancer cells was associated with enhanced migratory and invasive ability.
To unambiguously elucidate the enhanced effect of Tiam1 on colorectal cancer metastasis, we performed in vivo metastasis assay by SOI. Mice were sacrificed six weeks later because the mice of HT29/Tiam1 group were moribund. Autopsy was performed and the incidence of metastasis in the liver, lung and other organs were determined by macroscopic and histological examination. In HT29/Tiam1 group, 100% (7/7) of mice developed peritoneal metastasis that appeared as numerous white nodules on the peritoneum and the abdominal organs. In HT29/mock group, only 29% (2/7) of animal had peritoneal metastasis. The incidence of hepatic metastasis in mice of HT29/Tiam1 group was 57.1% (4/7), and two of them were easily detected by macroscopic observation and two were found by histological examination. HT29/mock group did not produce detectable tumors in livers. In addition, one of seven mice of HT29/Tiam1 group had tumor formation in the lung, speen, lymph node besides in the liver.
4. Building of whole-body visualizing orthotopic animal model in colon cancer
SW480/EGFP~+ cells stably expressed high-levels of enhanced green fluorescent protein (EGFP) and EGFP had no effect on cell proliferation. Visualizing orthotopic animal model developed with surgical orthotopic implantation (SOI) was built with 100% survival rate. After SOI, no animal had metastasis within 2 weeks, and subsequently, 6 weeks after SOI, 75% of animal had implantation metastasis and 50% of animal had liver metastasis. The whole-body visualizing orthotopic animal model was successfully validated by pathological detection.
5. Down-regulation of Tiam1 expression by RNAi
We constructed plasmids that expressed short hairpin RNAs that were targeted against Tiam1 under the control of the U6 promoter by lentiviral vector. Four different sequences were originally selected for targeting the Tiam1 gene. We infected SW480/EGFP~+ cells with lentival vectors and examined Tiam1 mRNA and protein expression and found site B vector was the most effective at blocking Tiam1 expression (73%). Then, SW480/EGFP~+ cells were infected with pLenti6/siTiam1 (site B) or pLenti6 (control vector), and incubated with blasticidin to select blasticidin-resistant single clones. We found clone 2 exhibited a dramatic knock down of Tiam 1 protein expression (88%) (Designated SW480/EGFP~+/Tiam1~-).
6. Effect of Tiam1 silencing on the biological behaviors of human CRC
SW480/EGFP~+/Tiam1~- cells showed a significantly reduced proliferation compared with SW480/EGFP~+/mock and SW480/EGFP~+ cells as determined by in vitro MTT assay. In addition, SW480/EGFP~+/Tiam1~- cells had a significant reduction in their ability to form colonies in plate as compared with SW480/EGFP~+/mock and SW480/EGFP~+ cells.
The result of in vitro invasion assay showed that SW480/EGFP~+/Tiam1~- cells had significantly reduced invasiveness as compared with SW480/EGFP~+/mock and SW480/EGFP~+ cells.
The effect of Tiam1 on in vivo tumor growth was assessed by subcutaneous injection of SW480/EGFP~+/Tiam1~- and SW480/EGFP~+/mock cells for thirty days. Compared with SW480/EGFP~+/mock cells, the silencing of Tiam1 led to a pronounced decrease in SW480/EGFP~+/Tiam1~- cells growth starting from day 15, up to 1.3 fold decrease in mice of tumor volume at day 30 after cell injection.
The metastatic ability evaluated by whole-body visualizing orthotopic animal model in SW480/EGFP~+/Tiam1~- cells was inhibited after Tiam1 silencing. In SW480/EGFP~+/mock group, 75% (6/8) of mice developed peritoneal metastasis, 37.5% (3/8) had hepatic metastasis, and 12.5% (1/8) had lung metastasis. In SW480/EGFP~+/Tiam1~- group, 37.5% (3/8) of mice had peritoneal metastasis and none of them had hepatic and lung metastasis.
7. Exploring the molecular mechanism underlying Tiam1-mediated multiple functions in CRC using gene microarray
Gene expression profiles of SW480/EGFP~+/Tiam1~- and SW480/EGFP~+/mock cells were obtained and the microarray findings were confirmed by Real Time PCR. We obtained 1026 of Tiam1-related genes which 647 genes were down-regulated and 379 gnes were up-regulated in SW480/EGFP~+/Tiam1~- cells. The function of these 647 differentially expressed genes involved in cell cycle, regulation of cell cycle, DNA replication, DNA metabolism and regulation of cellular physiological process. The pathway of these differentially expressed genes included wnt signalling pathway, p53 pathway, apoptosis signalling pathway, integrin signalling pathway, PI3 kanase pathway and Ras pathway.
8. Exploring the molecular mechanism underlying Tiam1-mediated multiple functions in CRC using proteomics analysis and peptide mass fingerprinting
Peoteomics represents a powerful approach to analyze alterations in protein expression and posttranslational modifications. To further understand the intracellular function of the Tiam1 in CRC, we implemented a proteomics-based approach to identify proteins associated with Tiam1 expression. The proteome of the HT29/Tiam1 and HT29/mock cells and of SW480/EGFP~+/Tiam1~- and SW480/EGFP~+/mock cells were compared using 2-D electrophoresis. Discrepancies in protein spots found and the significance of protein changes was evaluated using Student's t-tests. A number of spots were found to be significantly altered (P＜0.05) after Tiam1 transfection or Tiam1 silencing. Discrepancies in protein spots were excised from the 2-D gels and subjected to trypsin digestion and MALDI-TOF-MS. Twenty spots were identified successfully. Up-regulated proteins in HT29/Tiam1 cells include Fascin, heat shock protein 27 (HSP27), AnnexinⅡ, high-mobility group box1 (HMGB1), Glutathione S-transferase omega 1 (GSTO1) and IMP dehydrogenase. And down-regulated proteins in HT29/Tiam1 cells include Retinal dehydrogenase 1, annexinⅣand 11s regulator. Down-regulated proteins in SW480/EGFP~+/Tiam1~- cells include high mobility group box 1 (HMGB1), 26S proteasome non-ATPase regulatory subunit 7(PSD7), LysophospholipaseⅠ(LYPA1), Mitochondrial 28S ribosomal protein S22(MRP-S22), COPD(delta-COP), Phosphoglycerate mutase 1(PGAM1). These results suggested that these differentially expressed proteins were involved in cellular metabolism, cell proliferation, cytoskeletal network and other cell processes.
9. transcriptome and integration with its proteome of Tiam1-related genes
The identity between transcriptome and proteome of Tiam1-related genes were systematically analysed and integrated. We obtained three genes, which HMGB1 and PGAM1 were up-regulated in HT29/Tiam1 cells and down-regulated in SW480/EGFP~+/Tiam1~- cells, while ANXA4 down-regulated in HT29/Tiam1 cells and up-regulated in SW480/EGFP~+/Tiam1~- cells.
CONCLUSION
1. Tiam1 gene plays an important role in proliferation, invasion and metastasis of CRC and is a metastasis-related gene. Clinically it may be a useful indicator of the tumor progression and metastasis in CRC.
2. Tiam1 may involve in many pathways to mediate proliferation, invasion and metastasis of CRC. And we found three genes, HMGB1, PGAM1 and ANXA4, have close relationship with Tiam1. These data will be helpful to elucidate the molecular mechanism of Timn1 and provide new clues.
结直肠癌是常见的恶性肿瘤之一，近十年来，结直肠癌在我国的发病率呈逐年上升的趋势。转移是影响患者治疗效果和死亡的主要原因，探讨与结直肠癌转移相关的因素，寻找预防和治疗的有效途径，是当代肿瘤学研究的一项迫切任务。
在我们的前期研究中，利用自行研制的含有447个已知肿瘤转移相关基因cDNA芯片，筛选出51个结直肠癌转移相关基因。应用文献轮廓挖掘微阵列表达数据的生物信息学方法，筛选出4个与结直肠癌转移密切相关的新基因，并对其中一个新的结直肠癌转移相关基因—T淋巴瘤侵袭转移诱导因子1(T lymphoma invasion and metastasis，Tiaml)，在RNA水平进行了初步验证。
Tiaml是鸟苷酸转换因子(guanine nucleotide exchange factors，GEFs)家族成员，GEFs的主要功能是调节Rho GTPase的活性。Rho GTPase家族包括Rho、Rac及Cdc42，是Ras超家族的成员。Tiaml体内外均可以特异性地激活Rho GTPase家族中的Racl。新近的研究发现Tiaml不但激活Rac，更重要的是Tiaml直接与胞浆和胞膜上的蛋白质相互作用，将它们耦合到Tiaml-Rac信号通路上，从而影响Rac信号通路的特异性。
Tiaml基因最初在小鼠T淋巴瘤细胞高侵袭变异株中分离鉴定。随后，在乳腺癌、肺癌及Ras诱导的皮肤癌等肿瘤证实Tiaml具有明显的促进肿瘤进展和转移的作用。在Tiaml敲除的动物模型中，小鼠皮肤癌发生率明显减低并且肿瘤进展缓慢，Malliri等认为Tiaml在Ras诱导皮肤癌发生的启动和进展阶段发挥关键作用，这一效应与Tiam1表达量有明显的关系。上述的研究表明，Tiam1是多种肿瘤的促癌基因和促进转移基因。
本研究重点探讨Tiam1基因对结直肠癌增殖、侵袭及转移的影响和作用机制，阐明Tiam1在结直肠癌转移中的主要生物学功能，为结直肠癌转移的早期诊断和治疗奠定理论基础。
方法
1．结直肠癌组织中Tiam1的表达及临床意义
应用免疫组化S-P法，检测157例结直肠癌和配对的30例正常结直肠黏膜、35例结直肠癌区域淋巴结转移癌以及32例结直肠腺瘤组织中Tiam1蛋白的表达。
2．Tiam1过表达对结直肠癌细胞生物学特性的影响
用RT-PCR方法检测Tiam1基因在9种结直肠癌细胞株中的表达，筛选Tiam1不表达的细胞株；将Tiam1／C1199HA cDNA导入内源性不表达Tiam1基因的人结直肠癌HT29细胞株中，G418筛选抗性单克隆，RT-PCR、免疫组化及Western blot鉴定转染后Tiam1基因在细胞中的表达；观察Tiam1基因转染前后，细胞增殖、体外侵袭、成瘤能力及体内转移能力的变化。
3．建立整体可视化结肠癌原位及转移动物模型
利用阳离子脂质体Lipofectamine~(TM)2000将pEGFP-N1质粒导入人结直肠癌SW480细胞，获得稳定表达绿色荧光蛋白(enhanced green fluorescent protein，EGFP)的细胞株SW480／EGFP~+；通过结肠癌原位手术移植的方法建立结肠癌原位及转移模型，利用肿瘤细胞表达EGFP的特点，借助整体荧光体视镜结合IPP5.0软件采集、分析EGFP发出的荧光信号，在动物活体实时观察结肠癌局部成瘤及转移情况；利用MTT法检测和分析EGFP对细胞增殖能力的影响；应用病理学方法对结肠癌原位及转移动物模型进行评价和鉴定。
4．Tiam1表达沉默对结直肠癌细胞生物学特性的影响
设计Tiam1干扰片段，构建含U6启动子的慢病毒载体，将慢病毒载体质粒与辅助质粒共转染293FT病毒包装细胞，收集病毒上清，进行病毒滴度测定，用慢病毒感染结直肠癌细胞，用RT-PCR和Western blot鉴定有效的干扰载体。
用含Tiam1特异性干扰片段的慢病毒感染SW480／EGFP~+细胞，用含空载体的病毒做对照。用杀稻瘟菌素筛选抗性克隆，挑取抗性单克隆，荧光定量PCR和Western blot鉴定干扰后Tiam1基因在细胞中的表达；利用MTT法、平板克隆形成实验检测Tiam1对细胞体外增殖的影响、体外侵袭实验检测Tiam1对结直肠癌侵袭能力的影响、在裸鼠体内观察Tiam1对结直肠癌细胞致瘤能力的影响及利用可视化结直肠癌原位动物模型观察Tiam1对结直肠癌细胞体内转移能力的影响。
5．利用基因芯片和比较蛋白质组学技术分析Tiam1在结直肠癌增殖、侵袭和转移中的作用
利用Affymetrix公司的寡核苷酸芯片(47，400个转录本，包含38，500个已知基因)检测Tiam1基因表达沉默后差异基因表达谱。应用Gene Ontology、MILANO、Panther、Genomatix等软件进行生物信息学分析。
利用蛋白质组学技术分离HT29／Tiam1和HT29／mock细胞、SW480／EGFP~+／Tiam1~-和SW480／EGFP~+／mock细胞的总蛋白，建立重复性和分辨率较好的细胞双向凝胶电泳图谱，应用基质辅助激光解吸电离飞行时间质谱鉴定差异表达的蛋白质点。利用软件过滤基线峰、识别信号峰，利用Matrixscience公司的在线软件Mascot Distiller进行搜索分析。
利用荧光定量PCR、Western blot方法对所获得的差异基因和／或差异蛋白质进行鉴定。
结果
1．结直肠癌组织中Tiam1蛋白的表达
免疫组化结果显示Tiam1在正常结直肠黏膜、腺瘤、癌和淋巴结转移癌四种类型的组织中表达明显不同，差异具有显著性(x~2=23.561，P＜0.01)，两两比较发现，腺瘤Tiam1表达比正常结直肠黏膜组织强(Z=-2.423，P＜0.05)，淋巴结转移癌组织Tiam1表达比结直肠癌组织强(Z=-2.051，P＜0.05)，而结直肠癌组织与腺瘤组织Tiam1表达差异无显著性(Z=-0.938，P＞0.05)。在结直肠癌组织中，伴发区域淋巴结转移的癌组织比未发生转移的癌组织Tiam1表达明显增强，差异具有显著性(Z=-3.176，P＜0.01)。研究结果提示，Tiam1蛋白表达与结直肠癌转移高度相关，即Tiam1表达弱或阴性者转移的发生率低，表达强者其转移发生率高。
2．Tiam1质粒的转染及建立稳定表达Tiam1的结直肠癌细胞株
应用RT-PCR检测9种结直肠癌细胞株Tiam1基因的表达，发现HT29细胞内源性不表达Tiam1基因。将Tiam1质粒导入HT29细胞中，挑取G418抗性单克隆，RT-PCR及Western blot确定Tiam1在转染克隆1中表达最强(命名为HT29／Tiam1)，Tiam1在空载体对照中不表达(命名为HT29／mock)。
3．Tiam1过表达对结直肠癌细胞生物学特性的影响
通过MTT法，我们检测了Tiam1对结直肠癌细胞体外增殖能力的影响，与HT29／mock细胞相比，HT29／Tiam1细胞的增殖能力明显增强(F=239.197，P＜0.01)。此外，接种HT29／Tiam1细胞的裸鼠皮下肿瘤生长明显加快(F=48.410，P＜0.01)，肿瘤体积自皮下注射后的第七天起出现显著性差异，接种HT29／Tiam1细胞的裸鼠皮下肿瘤第20天的平均体积是HT29／mock细胞组的2.5倍。进行结直肠癌细胞外科原位移植6周后，接种HT29／Tiam1细胞的裸鼠结肠原位肿瘤重量明显大于接种HT29／mock细胞的裸鼠(t=-14.916，P＜0.01)。
体外侵袭实验证明，肿瘤细胞侵袭实验24h后，HT29／mock和HT29／Tiam1细胞均穿透基底膜向下侵袭，在聚碳酸酯膜上可见变形的肿瘤细胞，HT29／Tiam1细胞侵袭能力是HT29／mock细胞的1.9倍，说明Tiam1基因的表达增强了结直肠癌细胞的侵袭能力。
采用外科原位移植的方法比较HT29／mock和HT29／Tiam1细胞的体内转移能力。在HT29／Tiam1细胞组，100％(7／7)裸鼠发生了腹腔转移、57.1％(4／7)发生了肝转移、其中有一只裸鼠发生了广泛转移，包括脾、肺及淋巴结等转移。在HT29／mock细胞组，只有29％(2／7)裸鼠发生了腹腔转移，没有一只裸鼠发生远处转移。
4．整体可视化结肠癌原位及转移动物模型观察
本研究建立的结直肠癌细胞株SW480／EGFP~+稳定、高效的表达绿色荧光蛋白，且增殖能力与SW480相比，没有显著性差异(F=1.171，P＞0.05)；采用外科手术原位移植的方法建立结肠癌可视化原位动物模型，成功率达到100％。移植2周后，结肠原位成瘤，未发现有转移情况，手术6周后，75％的裸鼠发生了腹腔转移，50％发生了肝转移。通过病理学方法，验证了可视化原位及转移动物模型的可靠性。
5．通过RNAi技术沉默结直肠癌细胞株Tiam1表达，建立Tiam1基因稳定沉默的结直肠癌细胞株
构建了四个干扰Tiam1基因表达的慢病毒干扰载体，将慢病毒质粒和辅助质粒共转染293FT病毒包装细胞，收获病毒上清，测定病毒滴度为7×10~5Transducing Unit(TU)／ml。利用慢病毒感染SW480／EGFP~+细胞，荧光定量PCR和Western blot结果发现含片段B的慢病毒干扰载体的干扰效率最高(73％)。利用含干扰片段B的慢病毒和空载体病毒感染SW480／EGFP~+细胞，挑取杀稻瘟菌素抗性单克隆，荧光定量PCR和Western blot结果发现Tiam1在干扰克隆2中表达降低最明显(命名为SW480／EGFP~+／Tiam1~-)，干扰效率达88％。
6．Tiam1基因表达沉默后对结直肠癌细胞生物学特性的影响
MTT法观察Tiam1基因表达沉默后体外细胞的增殖情况，与SW480／EGFP~+／mock和SW480／EGFP~+细胞相比，SW480／EGFP~+／Tiam1~-细胞的增殖速度明显减慢，并且呈时间依赖关系(F=3.361，P＜0.01)。平板克隆形成实验显示SW480／EGFP~+／Tiam1~-细胞的活力显著下降，差异有统计学意义(F=7.244，P＜0.05)。这些结果均说明Tiam1表达水平减低后，肿瘤细胞体外生长被显著抑制。
体外侵袭小室检测Tiam1基因表达沉默后细胞侵袭能力的改变，结果显示，与SW480／EGFP~+／mock和SW480／EGFP~+细胞相比，SW480／EGFP~+／Tiam1~-细胞的侵袭能力明显降低，差异具有显著性(F=45.645，P＜0.01)，说明Tiam1表达沉默明显抑制了结直肠癌细胞的体外侵袭能力。
通过结直肠癌可视化动物模型，我们观察了Tiam1基因表达沉默后对结直肠癌增殖和转移能力的影响。将结直肠癌细胞接种于裸鼠的皮下，通过30天连续的观察，我们发现Tiam1基因表达沉默后，显著性地抑制了结直肠癌细胞的体内增殖能力(F=2.256，P＜0.05)，增殖差异从第15天开始，到第30天，差异达到1.3倍。
将Tiam1表达沉默的细胞接种于裸鼠结肠原位后，细胞的转移能力受到了限制。在SW480／EGFP~+／mock组，75％(6／8)裸鼠发生了腹腔转移，37.5％(3／8)裸鼠出现肝转移，12.5％(1／8)形成肺转移，而在SW480／EGFP~+／Tiam1~-组只有37.5％(3／8)形成腹腔转移，未发现裸鼠形成远处转移。
7．利用基因芯片技术探讨Tiam1在结直肠癌增殖、侵袭、转移中的作用
利用Affymetrix公司的HG-U133 plus 2寡核苷酸芯片对SW480／EGFP~+／Tiam1~-和SW480／EGFP~+／mock细胞的基因表达谱进行分析，获得Tiam1相关基因1026个，包括与Tiam1作用协同的基因647个，作用拮抗的基因379个。对获得的基因进行功能分类发现，与Tiam1基因协同作用的基因主要涉及了细胞周期、细胞周期调控、DNA复制、核苷酸代谢、调节细胞生理过程等，与Tiam1基因拮抗作用的基因主要涉及了生物过程负调控、抑制酶活性、细胞过程的负调控基因等。对获得的基因进行信号通路分类发现，参与Tiam1调节作用的基因主要涉及了Wnt通路、p53通路、凋亡信号通路、整合素信号通路、P13K信号通路、Ras信号通路等。
8．利用比较蛋白质组学技术分析Tiam1在结直肠癌增殖、侵袭、转移中的功能
比较蛋白质组学是当前蛋白质组学研究的主要策略，通过比较细胞在基因转染前后／沉默前后蛋白质表达差异，发现与目的基因作用相关的蛋白质。在本研究中，与HT29／mock细胞相比，HT29／Tiam1细胞表达的蛋白质在数量和表达水平上发生了一定变化，而这些发生改变的蛋白质与Tiam1的促进转移作用相关；SW480／EGFP~+／Tiam1~-细胞为通过RNAi技术使Tiam1表达沉默的细胞，其表达的蛋白质与SW480／EGFP~+／mock细胞相比，在数量和表达水平上亦发生了一定的变化，而这些发生改变的蛋白质亦与Tiam1的促进转移作用相关。因此，利用比较蛋白质组学的方法，分析这些细胞蛋白质表达的变化，可发现与Tiam1作用相关的蛋白质，从而了解其发挥作用的分子机制。在本研究中，利用蛋白质组学技术分离细胞的总蛋白，结合图像分析和基质辅助激光解析电离飞行时间质谱对部分差异表达的蛋白质点进行鉴定，共鉴定了20个差异表达的蛋白质点。我们对鉴定的蛋白质的功能进行分类，主要为与细胞代谢、细胞增殖、细胞骨架结构相关的蛋白，以及分子伴侣和未知功能的蛋白。这些差异蛋白质中有一些同肿瘤的增殖、转移密切相关。
9．Tiam1差异基因和／或蛋白质筛选过程中的转录组和蛋白质组的对接
在本研究中，我们利用基因芯片和比较蛋白质组学技术筛选与Tiam1作用的基因和／或蛋白质，转录组和蛋白质组对接的基因／蛋白质有3个，2个是与Tiam1基因功能协同的高迁移率族蛋白B1(high motility group box 1，HMGB1)和磷酸甘油酸变位酶(phosphoglycerate mutase，PGAM1)，他们随着Tiam1基因的表达上调而上调，表达沉默而下调；另一个是与Tiam1基因功能拮抗的膜联蛋白4(annexin A4，ANXA4)，它随着Tiam1基因的表达上调而下调，表达沉默而上调。
结论
1．Tiam1基因是结直肠癌增殖、侵袭、转移的促进基因；
2．Tiam1可能参与多条信号通路来介导它在结直肠癌中的作用，在相互作用的基因中HMGB1、PGAM1和ANXA4与Tiam1关系密切，他们是Tiam1信号通路中的新基因。本研究的创新之处
1．建立了整体可视化结直肠癌原位及转移动物模型，为结直肠癌的实验研究提供了理想的实验工具；
2．利用慢病毒载体和RNAi技术建立了稳定的Tiam1基因表达沉默的结直肠癌细胞株及Tiam1基因表达沉默的结直肠癌原位动物模型，为结直肠癌转移机制研究提供实验手段；
3．将芯片技术、比较蛋白质组学技术相结合，实现了转录组和蛋白质组的对接；
4．将RNAi技术、基因芯片技术、蛋白质组学技术相结合，用于Tiam1基因的功能研究，为阐明Tiam1在肿瘤转移中的作用提供新的证据，并综合分析Tiam1的信号转导通路，发现3个参与Tiam1信号通路新的成员。
BACKGROUND & OBJECTIVEColorectal cancer (CRC) is the second worldwide leading cause of cancer death in the world. The incidence rate of CRC in china is increasing fast during the past decades. Metastasis is one of the basic characteristic of malignant tumors and is the main cause which affects the therapeutic efficacy and leads to the death of cancer patients. It is an urgent task to work out the metastasis-associated factors and find out the preventive and therapeutic methods.In our previous studies, we prepared a cDNA microarray consisting of 447 tumor metastasis-associated candidate genes to screen metastasis-associated genes and obtained 51 candidate genes closely related to metastasis of colorectal cancer, four genes of which were selected as candidate metastatic genes of colorectal cancer by literature mining. Tiam1, one of the four candidate metastatic genes, was validated in the RNA level.Tiam1 is one of guanine nucleotide exchange factors (GEFs), which activate GTPases by promoting the exchange of their inactive GDP-bound forms to their active GTP-bound forms. Whereas Tiam1 displays GEF activity towards all three Rho-like GTPases Rac1, Cdc42 and RhoA in vitro, Tiam1 specifically activates Rac in vivo. Recent evidence suggests that Tiam1 could influence Rac GTPases signaling specificity in addition to promoting their activation. Tiam1 has been implicated to directly bind to many different cytoplasmic and membrane-associated proteins, which couples Tiam1-Rac activity to specific signaling pathways.
Tiam1 was originally identified as the invasion- and metastasis-inducing gene by proviral tagging in combination with in vitro selection for invasiveness in T lymphoma cells. The role of Tiam 1 in cellular migration, invasion and metastasis may not be limited to T lymphoma. It was reported to be important in promoting the tumor progression in a variety of cancers such as breast cancer, lung cancer and Ras-induced skin tumors. The possible role of Tiam1 in the metastasis of colorectal cancer is unclear.
In this study, we aim to clarify the possible role of Tiam1 gene in the proliferation, invasion and metastasis of CRC. It will be helpful to understand the molecular basis of CRC, and establish Tiam1 as a new target for early metastatic diagnostic markers and novel therapeutic strategies.
METHODS
1. Expression of Tiam1 in colorectal carcinomas and its clinical significance
The expression of Tiam1 was examined in specimens of 157 CRC, 30 paired nomal colorectal mucosae, 35 regional lymphatic metastases and 32 adenoma by immunohistochemical (S-P) method.
2. Effect of Tiam1 overexpression on the biological behaviors of human CRC
The expressions of Tiam1 gene in nine colorectal carcinoma cell lines were detected by reverse transcriptase polymerase chain reaction (RT-PCR). Tiam1/C1199HA cDNA was transfected into HT29, which had no endogenous Tiam1 expression. The Tiam1 expression in transfectant was determined by RT-PCR, immunohistochemistry and Western blot. The biological behaviors of tranfectant were investigated by MTT assay, in vitro invasion assay, tumorigenesis, and in vivo metastasis assay by surgical orthotopic transplantation (SOI).
3. Building the visualizing orthotopic animal model and observe real-time tumor growing and metastasis in colon cancer
The pEGFP-N1 plasmid was transfected into human colon carcinoma cell line SW480 to establish SW480/EGFP~+ cell line. MTT assay was engineered to detect the effect of EGFP on cell proliferation. The SW480/EGFP~+ cells were surgically orthotopically implanted as tissue fragments in the body of the colon of nude mice. Emitted fluorescence was collected and imaged through LT-9MACIMSYSPULS system and images were analyzed with IPP5.0 software. Whole-body optical images visualized real-time primary tumor growth and formation of metastatic lesions. The evaluation of orthotopic animal model was carried out with traditionally pathological methods.
4. Lentivirus-mediated silencing of Tiam1 gene in human CRC cell line
We transfected human CRC cell lines, SW480/EGFP~+ with Tiam1 specific RNAi lentiviral vectors as well as controls. Tiam1 silenced cells were screened by blasticidin. Quantitative RT-PCR and Western blot analysis were used to detect the expressions of Tiam1 mRNA and protein, respectively. MTT assay, plate colony formation assay, in vitro invasive assay, tumorigenesis, metastasis assay in vivo by whole-body visualizing orthotopic animal model of CRC were used to assess the functional effects of Tiam1 silencing on tumor cell proliferation, invasion and metastasis.
5. Exploring the possible molecular mechanism underlying Tiara1-mediated multiple functions in CRC using gene microarray and comparative proteomics method
Oligonucleotide microarray analysis was used to detect differences in gene expression between Tiam1-silencing SW480/EGFP~+ cells and the control.
Comparative two-dimensional gel electrophoresis (2-DE) technology was performed to separate the proteins between HT29/Tiam1 and HT29/mock cells and between SW480/EGFP~+/Tiam1~- and SW480/EGFP~+/mock cells respectively. Differentially expressed proteins were identified by Matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF-MS).
RESULTS
The main results and findings are as follows:
1. Expression of Tiam1 in CRC
Tiam1 was expressed heterogeneously among the normal colorectal mucosae, adenoma, carcinomas and lymphatic metastasis of colorectal carcinoma (x~2=23.561, P＜0.01). Tiam1 was higher expressed in adenoma compared with normal colorectal epithelium (Z=-2.423, P＜0.05). Tiam1 expression was significantly stronger in lymphatic metastasis in comparison with colorectal carcinoma (Z=-2.051, P＜0.05). Furthermore, the Tiam1 expression in colorectal carcinoma with lymph node metastasis is higher than that in colorectal carcinoma without lymph node metastasis (Z=-3.176, P＜0.01).
2. Transfection of C1199Tiam1 cDNA and selection of stable clones
To address the function of Tiam1 in CRC, we established stably transfected HT29 cells that overexpress Tiam1. As revealed by RT-PCR analysis, a higher level of Tiam1 expression was seen in the Tiam1 transfected clonel but not in the vector-only transfected clonel. The western blotting analysis also showed that Tiam1 expression in Tiam1 transfected clonel (designated HT29/Tiam1) was positive and vector-only transfected clonel (designated HT29/mock) was negative.
3. Effect of Tiam1 overexpression on the biological behaviors of human CRC
HT29/Tiam1 cells showed a significantly enhanced proliferation compared with the HT29/mock cells as determined by in vitro MTT assay. Next, the effect of Tiam1 on in vivo tumor growth was assessed by subcutaneous injection of HY29/Tiam1 and HT29/mock cells for twenty days. Compared with HT29/mock cells, the expression of Tiam1 led to a pronounced increase in HT29/Tiam1 cell growth starting from day 7, up to 2.5 fold increase in mice of tumor volume at day 20 after cell injection. Primary tumor growth after in vivo orthotopic implantation was also tested. In this assay, mice were sacrificed six weeks later, and tumors from surgical orthotopic implantation excised and weighed in HT29/Tiam1 group and HT29/mock group respectively. The result showed that Tiam1 expression led to a significant enhancement in orthotopic tumor growth.
Invasion through the extracellular matrix (ECM) is an important step in tumor metastasis. The ECMatrix served as a reconstituted basement membrane matrix of proteins. The number of cells migrating the ECMatrix was counted and the result showed that the HT29/Tiam1 cells posses a remarkable increase in invasiveness as compared with HT29/mock cells (P＜0.01). A 1.9-fold increase in the number of HT29/Tiam1 cells with an invasive ability was observed. These data indicated that Tiam1 expression in colorectal cancer cells was associated with enhanced migratory and invasive ability.
To unambiguously elucidate the enhanced effect of Tiam1 on colorectal cancer metastasis, we performed in vivo metastasis assay by SOI. Mice were sacrificed six weeks later because the mice of HT29/Tiam1 group were moribund. Autopsy was performed and the incidence of metastasis in the liver, lung and other organs were determined by macroscopic and histological examination. In HT29/Tiam1 group, 100% (7/7) of mice developed peritoneal metastasis that appeared as numerous white nodules on the peritoneum and the abdominal organs. In HT29/mock group, only 29% (2/7) of animal had peritoneal metastasis. The incidence of hepatic metastasis in mice of HT29/Tiam1 group was 57.1% (4/7), and two of them were easily detected by macroscopic observation and two were found by histological examination. HT29/mock group did not produce detectable tumors in livers. In addition, one of seven mice of HT29/Tiam1 group had tumor formation in the lung, speen, lymph node besides in the liver.
4. Building of whole-body visualizing orthotopic animal model in colon cancer
SW480/EGFP~+ cells stably expressed high-levels of enhanced green fluorescent protein (EGFP) and EGFP had no effect on cell proliferation. Visualizing orthotopic animal model developed with surgical orthotopic implantation (SOI) was built with 100% survival rate. After SOI, no animal had metastasis within 2 weeks, and subsequently, 6 weeks after SOI, 75% of animal had implantation metastasis and 50% of animal had liver metastasis. The whole-body visualizing orthotopic animal model was successfully validated by pathological detection.
5. Down-regulation of Tiam1 expression by RNAi
We constructed plasmids that expressed short hairpin RNAs that were targeted against Tiam1 under the control of the U6 promoter by lentiviral vector. Four different sequences were originally selected for targeting the Tiam1 gene. We infected SW480/EGFP~+ cells with lentival vectors and examined Tiam1 mRNA and protein expression and found site B vector was the most effective at blocking Tiam1 expression (73%). Then, SW480/EGFP~+ cells were infected with pLenti6/siTiam1 (site B) or pLenti6 (control vector), and incubated with blasticidin to select blasticidin-resistant single clones. We found clone 2 exhibited a dramatic knock down of Tiam 1 protein expression (88%) (Designated SW480/EGFP~+/Tiam1~-).
6. Effect of Tiam1 silencing on the biological behaviors of human CRC
SW480/EGFP~+/Tiam1~- cells showed a significantly reduced proliferation compared with SW480/EGFP~+/mock and SW480/EGFP~+ cells as determined by in vitro MTT assay. In addition, SW480/EGFP~+/Tiam1~- cells had a significant reduction in their ability to form colonies in plate as compared with SW480/EGFP~+/mock and SW480/EGFP~+ cells.
The result of in vitro invasion assay showed that SW480/EGFP~+/Tiam1~- cells had significantly reduced invasiveness as compared with SW480/EGFP~+/mock and SW480/EGFP~+ cells.
The effect of Tiam1 on in vivo tumor growth was assessed by subcutaneous injection of SW480/EGFP~+/Tiam1~- and SW480/EGFP~+/mock cells for thirty days. Compared with SW480/EGFP~+/mock cells, the silencing of Tiam1 led to a pronounced decrease in SW480/EGFP~+/Tiam1~- cells growth starting from day 15, up to 1.3 fold decrease in mice of tumor volume at day 30 after cell injection.
The metastatic ability evaluated by whole-body visualizing orthotopic animal model in SW480/EGFP~+/Tiam1~- cells was inhibited after Tiam1 silencing. In SW480/EGFP~+/mock group, 75% (6/8) of mice developed peritoneal metastasis, 37.5% (3/8) had hepatic metastasis, and 12.5% (1/8) had lung metastasis. In SW480/EGFP~+/Tiam1~- group, 37.5% (3/8) of mice had peritoneal metastasis and none of them had hepatic and lung metastasis.
7. Exploring the molecular mechanism underlying Tiam1-mediated multiple functions in CRC using gene microarray
Gene expression profiles of SW480/EGFP~+/Tiam1~- and SW480/EGFP~+/mock cells were obtained and the microarray findings were confirmed by Real Time PCR. We obtained 1026 of Tiam1-related genes which 647 genes were down-regulated and 379 gnes were up-regulated in SW480/EGFP~+/Tiam1~- cells. The function of these 647 differentially expressed genes involved in cell cycle, regulation of cell cycle, DNA replication, DNA metabolism and regulation of cellular physiological process. The pathway of these differentially expressed genes included wnt signalling pathway, p53 pathway, apoptosis signalling pathway, integrin signalling pathway, PI3 kanase pathway and Ras pathway.
8. Exploring the molecular mechanism underlying Tiam1-mediated multiple functions in CRC using proteomics analysis and peptide mass fingerprinting
Peoteomics represents a powerful approach to analyze alterations in protein expression and posttranslational modifications. To further understand the intracellular function of the Tiam1 in CRC, we implemented a proteomics-based approach to identify proteins associated with Tiam1 expression. The proteome of the HT29/Tiam1 and HT29/mock cells and of SW480/EGFP~+/Tiam1~- and SW480/EGFP~+/mock cells were compared using 2-D electrophoresis. Discrepancies in protein spots found and the significance of protein changes was evaluated using Student's t-tests. A number of spots were found to be significantly altered (P＜0.05) after Tiam1 transfection or Tiam1 silencing. Discrepancies in protein spots were excised from the 2-D gels and subjected to trypsin digestion and MALDI-TOF-MS. Twenty spots were identified successfully. Up-regulated proteins in HT29/Tiam1 cells include Fascin, heat shock protein 27 (HSP27), AnnexinⅡ, high-mobility group box1 (HMGB1), Glutathione S-transferase omega 1 (GSTO1) and IMP dehydrogenase. And down-regulated proteins in HT29/Tiam1 cells include Retinal dehydrogenase 1, annexinⅣand 11s regulator. Down-regulated proteins in SW480/EGFP~+/Tiam1~- cells include high mobility group box 1 (HMGB1), 26S proteasome non-ATPase regulatory subunit 7(PSD7), LysophospholipaseⅠ(LYPA1), Mitochondrial 28S ribosomal protein S22(MRP-S22), COPD(delta-COP), Phosphoglycerate mutase 1(PGAM1). These results suggested that these differentially expressed proteins were involved in cellular metabolism, cell proliferation, cytoskeletal network and other cell processes.
9. transcriptome and integration with its proteome of Tiam1-related genes
The identity between transcriptome and proteome of Tiam1-related genes were systematically analysed and integrated. We obtained three genes, which HMGB1 and PGAM1 were up-regulated in HT29/Tiam1 cells and down-regulated in SW480/EGFP~+/Tiam1~- cells, while ANXA4 down-regulated in HT29/Tiam1 cells and up-regulated in SW480/EGFP~+/Tiam1~- cells.
CONCLUSION
1. Tiam1 gene plays an important role in proliferation, invasion and metastasis of CRC and is a metastasis-related gene. Clinically it may be a useful indicator of the tumor progression and metastasis in CRC.
2. Tiam1 may involve in many pathways to mediate proliferation, invasion and metastasis of CRC. And we found three genes, HMGB1, PGAM1 and ANXA4, have close relationship with Tiam1. These data will be helpful to elucidate the molecular mechanism of Timn1 and provide new clues.
引文
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