Tiam1基因在肝细胞癌侵袭转移中的功能研究
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摘要
研究背景和目的
原发性肝癌(Hepatocellular carcinoma,HCC,简称肝癌)是常见的恶性肿瘤之一,近十年来,肝癌在我国的发病率呈逐年上升的趋势。虽然随着诊疗水平的不断提高和放化疗方案的不断更新,肝癌患者的疗效日益显著,但其生存率并未显著提高,其主要原因是很多肝癌患者在行根治术前已出现了微转移。因此,探讨与肝癌转移相关的因素,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。
T淋巴瘤侵袭转移诱导因子1(T lymphoma invasion and metastasis 1,Tiam1)基因是Db1家族的鸟嘌呤核酸转换因子(guanine nucleotide exchange factor, GEFs)之一,GEFs主要功能是调节Rho三磷酸鸟嘌呤核苷酸水解酶(Rho GTPases)家族活性,是普遍存在的二磷酸鸟嘌呤核苷酸(GDP)与三磷酸鸟嘌呤核苷酸(GTP)转换因子,通过参与Tiam1-Rac信号传递通路调节细胞骨架重组、细胞周期进程、基因转录、细胞迁移和粘附等细胞生命活动,在肿瘤增殖、侵袭和转移过程中发挥重要作用。早期研究表明Tiam1是诱导人类T细胞淋巴瘤侵袭和转移的基因。
Tiam1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Tiam1具有明显的促进肿瘤进展和转移的作用。在Tiam1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Tiam1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转移基因。
本课题前期已完成如下研究工作：应用白行设计的包括部分正常组织和常见恶性肿瘤组织的组织芯片,采用免疫组化方法检测和观察人体正常组织和常见恶性肿瘤组织,213例临床随访资料齐全的肝癌组织和9种人肝癌细胞株及1种人正常肝细胞株中Tiam1蛋白的表达情况,发现Tiam1蛋白在多种肿瘤组织中高表达,表明Tiam1可能是导致肿瘤发生发展的重要分子；在肝癌组织中高表达,在肝硬化组织中低表达,而在正常肝组织中不表达,提示Tiam1可作为临床肝癌检测的重要肿瘤标志物。分析Tiam1蛋白在肝癌中的表达及其与各临床病理参数之间的关系,同时结合213例临床随访资料回顾性分析Tiam1蛋白表达与肝癌患者生存期的关系,表明Tiam1与患者预后、转移密切相关,提示Tiam1可作为肝癌预后判断的重要指标,对判断肝癌预后有重要意义；为肝癌患者的诊断、预后评价、综合治疗方案设计提供有力的理论依据。
在前期研究工作的基础之上,本研究进一步探讨Tiam1基因对肝癌增殖、侵袭及转移的影响和作用机制,拟应用慢病毒介导的基因沉默技术和基因转染技术作用于同一遗传背景、不同转移潜能的两种肝癌细胞株HCCLM6和MHCC97L,双向阐明Tiam1在肝癌增殖、侵袭、转移中的主要作用,探讨Tiam1作为肝癌分子靶点的可行性,为肝癌患者的早期诊断、预后评价和综合治疗奠定理论基础。
方法
1.Tiaml基因／蛋白在人正常肝细胞和肝癌细胞中的表达及临床意义
用荧光定量PCR. Western blot、免疫细胞化学、免疫细胞荧光、免疫细胞荧光共聚焦等方法检测Tiam1基因／蛋白在高转移潜能肝癌细胞株HCCLM6(简称M6)、低转移潜能肝癌细胞株MHCC97L(简称97L)和1株正常肝细胞株HL-7702中的表达,验证高低转移潜能肝癌细胞株中Tiam1的表达情况。
2. Tiaml过表达对肝癌细胞生物学特性的影响
对Tiaml/C1199HA cDNA质粒进行全长测序鉴定后,利用SBI慢病毒载体构建系统,按照SBI过表达慢病毒包装试剂盒(System Biosciences公司)的操作说明进行慢病毒的包装和滴度测定。用包装后的慢病毒过表达载体感染97L细胞,绿色荧光蛋白挑取单克隆,荧光定量PCR、Western blot及免疫细胞荧光共聚焦等鉴定转染后Tiam1基因／蛋白在细胞中的表达；利用MTT法、平板克隆形成实验、细胞周期检测Tiam1对细胞体外增殖能力的影响；细胞划痕和体外侵袭实验检测Tiam1对肝癌细胞迁移能力和侵袭能力的影响；裸鼠皮下瘤模型观察Tiam1对肝癌细胞体内肿瘤增殖能力的影响；利用裸鼠尾静脉转移瘤模型观察Tiam1对肝癌细胞癌体内转移能力的影响。
3. Tiaml表达沉默对肝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,用慢病毒感染肝癌细胞M6,荧光定量PCR和Western blot鉴定有效的干扰载体。选取最合适的干扰片段继续实验,绿色荧光蛋白挑取单克隆,荧光定量PCR、Western blot及免疫细胞荧光共聚焦等鉴定干扰后Tiam1基因／蛋白在M6细胞中的表达；利用MTT法、平板克隆、流式细胞周期检测Tiam1沉默后对M6细胞体外增殖能力的影响；细胞划痕和体外侵袭实验检测Tiam1对肝癌细胞迁移能力和侵袭能力的影响；裸鼠皮下瘤模型观察Tiam1对肝癌细胞体内成瘤增殖能力的影响；利用裸鼠尾静脉转移瘤模型观察Tiam1对肝癌细胞体内转移能力的影响。
4.统计学处理
用SPSS 16.0软件包进行数据分析,采用重复测量方差分析对比转染前后和干扰前后肝癌细胞株的MTT体外增殖实验、细胞划痕实验、皮下成瘤实验是否具有显著性差异；采用单因素方差分析对比转染前后和干扰前后肝癌细胞株的荧光定量PCR、平板克隆形成实验、流式细胞术细胞周期实验、Transwell侵袭实验是否具有显著性差异；多重比较采用LSD法；P<0.05为有统计学意义。
结果
1.Tiam1基因在肝癌细胞株和正常细胞株中的表达情况
采用荧光定量PCR法检测2种人肝癌细胞株和1种人正常肝细胞株中Tiam1基因的表达情况。单因素方差分析结果表明：与正常肝细胞株HL-7702相比,Tiam1基因在M6细胞高表达,差异具有显著性(P=0.000),在97L细胞低表达,差异无显著性(P=0.766);Western blot.免疫细胞化学、免疫细胞荧光、免疫细胞荧光共聚焦结果均表明Tiam1在HL-7702和97L中低表达,在M6中高表达。
2.稳定特异性表达Tiam1基因对肝癌细胞生物学特性的影响
成功构建了以绿色荧光蛋白为报告基因的Tiam1过表达载体pCDF1-Tiam1+-copGFP,进行慢病毒的包装和滴度测定后将该载体转染至低转移潜能的肝癌细胞株97L中,并设立空白对照,绿色荧光为标记物挑取3个阳性单克隆,荧光定量PCR.Western blot结果发现单克隆97L／T2转染效率最高；将其命名为97L/copGFP/Tiam 1+,同时空白对照细胞株命名为97L/copGFP/mock,未经任何处理的细胞为97L。
MTT法观察稳定表达Tiam1基因后体外细胞的增殖情况,不同细胞在不同时间增殖速度有显著性差异,并呈时间依赖关系(F=24.658,P=0.000)；平板克隆形成实验结果也显示97L/copGFP/Tiam 1+细胞的活力明显较其他两组强(P=0.028,P=0.044),流式细胞术结果显示97L/copGFP/Tiam 1+细胞中S期比例明显较其他两组增加(P=0.000,P=0.000),这些结果均说明Tiam1表达水平增加后,显著增强肿瘤细胞体外生长能力。
细胞划痕实验和体外侵袭小室检测稳定表达特异性Tiam1基因后细胞迁移和侵袭能力的改变,结果显示不同细胞在不同时间迁移细胞数有显著性差异,并呈时间依赖关系(F=93.331,P=0.000)；与97L/copGFP/mock细胞和97L细胞相比,97L/copGFP/Tiam1+细胞的侵袭能力明显增加,差异具有统计学意义(P=0.000,P=0.000)。这些结果表明Tiam1表达水平增加显著增强肝癌细胞迁移和侵袭能力。
通过肝癌可视化动物模型,我们观察稳定表达Tiam1基因后对肝癌体内增殖和转移能力的影响。将97L/copGFP/mock细胞和97L/copGFP/Tiam1+细胞接种于裸鼠双侧背部皮下,通过30天连续的观察,我们发现特异性表达Tiam1基因后,显著地增加了肝癌细胞的体内增殖能力(F=192.294,P=0.000),增殖差异从第15天开始,到第30天,差异达到最大,为2.1倍。采用尾静脉注射法建立了稳定表达Tiam1前后肝癌细胞的裸鼠体内静脉转移瘤模型。2个月后处死裸鼠并在整体荧光体视镜下观察发现在97L/copGFP/mock组的6只裸鼠中仅有1只可观察到微小肺转移；而97L/copGFP/Tiam1+组的6只裸鼠中有3只出现了肺转移,3只出现肝转移,2只出现骨转移；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肝癌细胞的转移。
3. Tiam1基因表达沉默对肝癌细胞生物学特性的影响
利用RNA干扰技术,成功构建了4个以绿色荧光蛋白为报告基因的Tiam1特异性的慢病毒干扰载体pGCSIL-EGFP,通过慢病毒介导的RNAi技术沉默具有高转移潜能的肝癌细胞株M6内源性Tiam1基因的表达,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(84.0%)利用含干扰片段B的慢病毒和空载体病毒感染M6细胞,绿色荧光挑选单克隆,荧光定量PCR和Western blot结果发现Tiam 1在干扰克隆M6／T2中表达最弱,干扰效率达(90.4%),以干扰克隆M6／T2作为Tiam1基因稳定沉默的肝癌细胞株,将其命名为为M6/EGFP/Tiam1-,同时空白对照细胞株命名为M6/EGFP/mock,未经任何处理的原始细胞为M6。
MTT法观察稳定表达Tiam1基因后体外细胞的增殖情况,不同细胞在不同时间增殖速度有显著性差异,并呈时间依赖关系(F=59.732,P=0.000)；平板克隆形成实验结果也显示M6/EGFP/Tiam1细胞的活力明显较其他两组减弱(P=0.000,P=0.000)；流式细胞术结果显示M6/EGFP/Tiam1细胞中S期比例明显较其他两组减小(P=0.000,P=0.000),这些结果均说明Tiam1表达水平减弱后,显著抑制了肿瘤细胞体外增殖能力。
细胞划痕实验和体外侵袭小室检测稳定沉默Tiam1基因后细胞迁移和侵袭能力的改变,结果显示不同细胞在不同时间迁移细胞数有显著性差异,并呈时间依赖关系(F=248.998,P=0.000)；与M6细胞和M6/EGFP/mock细胞相比,M6/EGFP/Tiam1细胞的侵袭能力明显减弱,差异具有统计学意义(P=0.000,P=0.000)。这些结果表明Tiam1表达水平降低显著抑制肝癌细胞迁移和侵袭能力。
通过肝癌可视化动物模型,我们观察稳定沉默Tiam1基因表达后对肝癌增殖和转移能力的影响。将M6/EGFP/mock细胞和M6/EGFP/Tiam1-细胞接种于裸鼠双侧背部皮下,通过30天连续的观察,我们发现特异性沉默Tiam1基因表达后,显著抑制了肝癌细胞的体内增殖能力(F=192.294,P=0.000),增殖差异从第15天开始,到第30天,差异达到最大,为2.2倍。采用尾静脉注射法建立了稳定沉默Tiam1表达前后肝癌细胞的裸鼠体内转移模型。2个月后处死裸鼠并在整体荧光体视镜下观察发现在M6/EGFP/mock组的6只裸鼠中有6只出现了肺转移,5只出现肝转移,6只出现骨转移；而M6/EGFP/Tiam1组的6只裸鼠中有1只可观察到肺转移,1只出现骨转移,未见肉眼可见肝转移；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肝癌的转移。
结论
1.利用慢病毒介导的稳定转染技术初步研究Tiam1基因在肝癌增殖、侵袭、转移中的作用,发现Tiam1基因是肝癌增殖、侵袭、转移的促进基因。
2.利用慢病毒介导的基因稳定沉默技术进一步验证Tiam1基因在肝癌增殖、侵袭、转移中的作用,从反面确证Tiam1基因是肝癌增殖、侵袭、转移的促进基因。
本研究的创新之处
1.本项目以慢病毒为载体,分别建立了稳定表达外源性Tiam1基因肝癌细胞株97L/copGFP/Tiam 1+和特异性Tiam1基因稳定沉默的肝癌细胞株M6/EGFP/Tiam 1-,为深入研究Tiam1基因的功能提供了有价值的工具。
2.通过对比观察表达Tiam1基因肝癌细胞株97L/copGFP/Tiam 1+和特异性Tiam1基因表达沉默肝癌细胞株M6/EGFP/Tiam1-细胞的体内、外生物学能力的改变,初步证实Tiam1基因与肝癌侵袭转移密切相关。
BACKGROUND & OBJECTIVE
Hepatocellular carcinoma (HCC) is one of the common cancers in the world. The incidence rate of HCC 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.
Tiam1(T lymphoma invasion and metastasis 1)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. 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 Tiaml 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.
The role of Tiaml 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 Tiaml in the metastasis of colorectal cancer is unclear.
In our previous study, by using tissue chip of normal tissues and common malignant tumors which was designed and produced by ourselves and immunohistochemistry, Tiaml protein expressions were detected in human normal tissues and common malignant tumors,213 cases of hepatocellular carcinoma tissues, 9 human hepatocellular carcinoma cell lines and 1 human normal hepatic cell. Tiaml protein was highly expressed in many tumors and it can be acted as one important tumor maker for tumorigenesis and development. Tiaml protein was up-regulated in hepatocellular carcinoma tissues, expressed lowly in liver sclerotic tissues and had no expression in normal hepatic tissue, indicating that Tiaml may be regarded as the important marker for clinical detection of HCC.Combined evaluation of Tiaml protein high expression and clinical follow-up data analysis showed that Tiam1 had close relationship with prognosis and metastasis of hepatocellular carcinoma patients.Tiam1 may act as the important marker for judging prognosis of HCC.
In this study, we aim to clarify the possible role of Tiaml gene in the proliferation, invasion and metastasis of HCC.It will be helpful to understand the molecular basis of HCC,and establish Tiaml as a new target for early metastatic diagnostic markers and novel therapeutic strategies.
METHODS
1.Expression of Tiaml gene/protein in human normal liver cells and liver cancer cells.
Real-time PCR, Western blot, Immunocytochemistry, Cellular immunofluoresce-nce and Cellular immunofluorescence confocal were used to examine the expressions of Tiaml in 2 hepatocellular carcinoma cell lines named M6,97L and 1 normal named HL-7702.
2.Effect of Tiaml overexpression on the biological behaviors of human HCC
Lentiviral vector pCDF1-Tiam1-copGFP was constructed by gene cloning. Recombinant lentivirus was harvested from 293FT cells cotransfected with the pPACKF1 Packaging Plasmid Mix.Tiam1/C1199HA cDNA was transfected into the 97L cell,which had lower Tiaml expression. Single transfectant cells clone were established 97L/copGFP/Tiaml+line by copGFP, Single mock cells clone were also established 97L/copGFP/mock line by copGFP. The Tiaml expression in transfectant was determined by RT-PCR, immunohistochemistry and Western blot. The biological behaviors of tranfectant were investigated by MTT assay, plate colony formation assay, flow cytometry, cell wounding heal assay, invasion assays in vitro, subcutaneous tumor model in nude mice, metastasis assay in vivo through the lateral tail-vein injection through LT-9MACIMSYSPULS system which were used to assess the functional effects of Tiaml silencing 97L cells in vitro or in vivo respectively.
3.Lentivirus-mediated silencing of Tiaml gene in human HCC M6 cell line.
Lentiviral expression vectors containing enhanced green fluorescence protein (EGFP) and Tiaml small interfering RNA (Lenti-Tiamlsi), or the control siRNA (Lenti-NC) gene were constructed. Human HCC M6 cell line was transfected with a different multiplicity of infection (MOI) of Lenti-Tiaml-si-A, B,C,D or Lenti-NC, and cultured to obtain stably-transfected M6/EGFP/Tiam1- and M6/EGFP/mock cells. The expression of Tiam1 mRNA/protein was determined by real-time PCR, western blot and confocal immunofluorescence.The biological behaviors of tranfectant were investigated by MTT assay, plate colony formation assay, flow cytometry, cell wounding heal assay, invasion assays in vitro, subcutaneous tumor model in nude mice, metastasis assay in vivo through the lateral tail-vein injection through LT-9MACIMSYSPULS system which were used to assess the functional effects of Tiam1 silencing M6 cells in vitro or in vivo, respectively.
4.Statistical analysis
Statistical analysis was done using SPSS 16.0. MTT assay, cell wounding heal assay, subcutaneous tumor model in nude mice were analyzed by repeated measure square analyze.Real-time PCR, plate colony formation assay, flow cytometry, invasion assays in vitro were analyzed one-factor analysis of variance.
RESULTS
The main results and findings are as follows:
1.The expressions of Tiaml gene in human normal liver cell line and liver cancer cell lines.
Tiam1 gene expressions in 2 human hepatocellular carcinoma cell lines and 1 human normal hepatic cell line were detected by real time PCR. The results of one-factor analysis of variance showed that the expressions of Tiam1 in 3 cell lines had significant difference (F=155.975,P=0.000).The expresions of Tiaml in M6 was higher than 97L and normal hepatic cell line HL-7702 with significant difference.
2.Effect of Tiam1 overexpression on the biological behaviors of human HCC
Recombinant pCDFl-Tiaml-copGFP lentivirus vector was constructed and identified by restriction endonuclease analysis and DNA sequencing.6771bp vector segment and 3800bp Tiam1 were got after EcoRI digested.After 60 hours of transferring pCDF1-Tiam1-copGFP and packaging plasmid into 293FT cell,the green fluorescence could be observed in some of the transferring cells.
97L cells were infected with recombinant lentivirus or mock lentivirus. A higher level of Tiaml expression was seen in the Tiaml transfected clone 2, we chose 97L/T2 clone (named 97L/copGFP/Tiam1+)cells and mock clone (named 97L/copGFP/mock) as function experiments in vitro or vivo.The result showed a significantly enhanced proliferation features compared with the 97L and 97L/copGFP/mock cells as determined by Real-time PCR, Western blot, Immunofluorescence confocal. The results showed that Tiaml overexpression promoted proliferation:MTT assay (F=24.658,P=0.000); plate colony formation assay(P=0.028,P=0.044); flow cytometry (P=0.000,P=0.000);cell wounding heal assay(F=93.331,P=0.000) and invasive capabilities(P=0.000,P=0.000)in 97L/copGFP/Tiaml+cells compared with 97L or 97L/copGFP/mock in vitro. Next, the effect of Tiaml on tumor growth was assessed by subcutaneous injection of 97L/copGFP/Tiam1+ and 97L/copGFP/mock cells for thirty days in vivo. Compared with 97L/copGFP/mock cells, the expression of Tiaml led to a pronounced increase in 97L/copGFP/Tiaml+cell growth starting from day 15,up to 2.1-fold increase in mice of tumor volume at day 30 after cell injection(F=192.294,P=0.000).To unambiguously elucidate the enhanced effect of Tiaml on HCC metastasis, we performed and metastasis assay in vivo through the lateral tail-vein injection. Mice were sacrificed 2 months later because the mice of 97L/copGFP/Tiaml+ group were moribund. The metastasis in the liver, lung, bone and other organs were determined the metastatic ability by whole-body visualizing orthotopic animal model and histological examination;in 97L/copGFP/Tiam1+ group,50%(3/6) of mice developed lung metastasis that appeared as numerous green nodules on the lung, 50%(3/6) of mice developed liver metastasis,34%(2/6) developed bone metastasis. In 97L/copGFP/mock group, only 29%(1/6) of animal had lung metastasis.
These data indicated that Tiaml expression in liver 97L cell was associated with enhanced migratory and invasive ability.
3.Effect of Tiaml silencing on the biological behaviors of human HCC M6 cells.To require prolonged suppression of Tiaml protein in M6 cells, we constructed 4 vshRNA (pGC-LV recombination vector) containing Tiam1 interfere sequens A, B, C and D.Real-time PCR analysis showed that the mRNA levels of Tiaml in four Tiaml-si cells were all suppressed, especially in Tiaml-si-B cells (84.0%).Then, we reinfected M6 cells.with si-B for further identifycation.
M6 cells were infected with recombinant lentivirus or mock lentivirus. A lower level of Tiaml expression was seen in the Tiaml transfected clone 2(90.4%), we chose M6/T2 clone (named M6/EGFP/Tiaml-)cells and mock clone (named M6/EGFP/mock) as function experiments in vitro or vivo.The result showed a significantly declined proliferation features of M6/EGFP/Tiam1- compared with the M6 and M6/EGFP/mock cells as determined by Real-time PCR, Western blot, Immunofluorescence confocal. The results showed that Tiam1 overexpression promoted proliferation:MTT assay (F=59.732,P=0.000);plate colony formation assay(P=0.028, P=0.044); flow cytometry (P=0.000, P=0.000); cell wounding heal assay(F=248.998,P=0.000) and invasion assays in vitro (P=0.000, P=0.000) in M6/EGFP/Tiam1- cells compared with M6 or M6/EGFP/mock in vitro.Next, the effect of Tiaml on tumor growth was assessed by subcutaneous injection of M6/EGFP/mock and M6/EGFP/Tiam1- cells for thirty days in vivo.Compared with M6/EGFP/mock cells, the silencing of Tiaml led to a pronounced decrease in M6/EGFP/Tiam1- cell growth starting from day 15,up to 2.2-fold decrease in mice of tumor volume at day 30 after cell injection(F=192.294,P=0.000). To unambiguously elucidate the declined effect of Tiaml on HCC metastasis, we performed and metastasis assay in vivo through the lateral tail-vein injection.Mice were sacrificed 2 months later because the mice of M6/EGFP/Tiam1- group were moribund.The metastasis in the liver, lung, bone and other organs were determined by the metastatic ability evaluated by whole-body visualizing orthotopic animal model and histological examination. In M6/EGFP/Tiam1- group, only 17%(1/6) of animal had lung metastasis,17%(1/6) had bone metastasis. But in M6/EGFP/mock group,100%(6/6) of mice developed lung metastasis that appeared as numerous green nodules on the lung,83%(5/6) of mice developed liver metastasis,100%(6/6) developed bone metastasis.
These data indicated that Tiam1 silencing in liver M6 cell was associated with decreasing migratory and invasive ability.
CONCLUSION
Tiaml gene plays an important role in proliferation, invasion and metastasis of HCC and is a metastasis-related gene.Clinically it may be a useful indicator of the tumor progression and metastasis in HCC.
原发性肝癌(Hepatocellular carcinoma,HCC,简称肝癌)是常见的恶性肿瘤之一,近十年来,肝癌在我国的发病率呈逐年上升的趋势。虽然随着诊疗水平的不断提高和放化疗方案的不断更新,肝癌患者的疗效日益显著,但其生存率并未显著提高,其主要原因是很多肝癌患者在行根治术前已出现了微转移。因此,探讨与肝癌转移相关的因素,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。
T淋巴瘤侵袭转移诱导因子1(T lymphoma invasion and metastasis 1,Tiam1)基因是Db1家族的鸟嘌呤核酸转换因子(guanine nucleotide exchange factor, GEFs)之一,GEFs主要功能是调节Rho三磷酸鸟嘌呤核苷酸水解酶(Rho GTPases)家族活性,是普遍存在的二磷酸鸟嘌呤核苷酸(GDP)与三磷酸鸟嘌呤核苷酸(GTP)转换因子,通过参与Tiam1-Rac信号传递通路调节细胞骨架重组、细胞周期进程、基因转录、细胞迁移和粘附等细胞生命活动,在肿瘤增殖、侵袭和转移过程中发挥重要作用。早期研究表明Tiam1是诱导人类T细胞淋巴瘤侵袭和转移的基因。
Tiam1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Tiam1具有明显的促进肿瘤进展和转移的作用。在Tiam1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Tiam1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转移基因。
本课题前期已完成如下研究工作：应用白行设计的包括部分正常组织和常见恶性肿瘤组织的组织芯片,采用免疫组化方法检测和观察人体正常组织和常见恶性肿瘤组织,213例临床随访资料齐全的肝癌组织和9种人肝癌细胞株及1种人正常肝细胞株中Tiam1蛋白的表达情况,发现Tiam1蛋白在多种肿瘤组织中高表达,表明Tiam1可能是导致肿瘤发生发展的重要分子；在肝癌组织中高表达,在肝硬化组织中低表达,而在正常肝组织中不表达,提示Tiam1可作为临床肝癌检测的重要肿瘤标志物。分析Tiam1蛋白在肝癌中的表达及其与各临床病理参数之间的关系,同时结合213例临床随访资料回顾性分析Tiam1蛋白表达与肝癌患者生存期的关系,表明Tiam1与患者预后、转移密切相关,提示Tiam1可作为肝癌预后判断的重要指标,对判断肝癌预后有重要意义；为肝癌患者的诊断、预后评价、综合治疗方案设计提供有力的理论依据。
在前期研究工作的基础之上,本研究进一步探讨Tiam1基因对肝癌增殖、侵袭及转移的影响和作用机制,拟应用慢病毒介导的基因沉默技术和基因转染技术作用于同一遗传背景、不同转移潜能的两种肝癌细胞株HCCLM6和MHCC97L,双向阐明Tiam1在肝癌增殖、侵袭、转移中的主要作用,探讨Tiam1作为肝癌分子靶点的可行性,为肝癌患者的早期诊断、预后评价和综合治疗奠定理论基础。
方法
1.Tiaml基因／蛋白在人正常肝细胞和肝癌细胞中的表达及临床意义
用荧光定量PCR. Western blot、免疫细胞化学、免疫细胞荧光、免疫细胞荧光共聚焦等方法检测Tiam1基因／蛋白在高转移潜能肝癌细胞株HCCLM6(简称M6)、低转移潜能肝癌细胞株MHCC97L(简称97L)和1株正常肝细胞株HL-7702中的表达,验证高低转移潜能肝癌细胞株中Tiam1的表达情况。
2. Tiaml过表达对肝癌细胞生物学特性的影响
对Tiaml/C1199HA cDNA质粒进行全长测序鉴定后,利用SBI慢病毒载体构建系统,按照SBI过表达慢病毒包装试剂盒(System Biosciences公司)的操作说明进行慢病毒的包装和滴度测定。用包装后的慢病毒过表达载体感染97L细胞,绿色荧光蛋白挑取单克隆,荧光定量PCR、Western blot及免疫细胞荧光共聚焦等鉴定转染后Tiam1基因／蛋白在细胞中的表达；利用MTT法、平板克隆形成实验、细胞周期检测Tiam1对细胞体外增殖能力的影响；细胞划痕和体外侵袭实验检测Tiam1对肝癌细胞迁移能力和侵袭能力的影响；裸鼠皮下瘤模型观察Tiam1对肝癌细胞体内肿瘤增殖能力的影响；利用裸鼠尾静脉转移瘤模型观察Tiam1对肝癌细胞癌体内转移能力的影响。
3. Tiaml表达沉默对肝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,用慢病毒感染肝癌细胞M6,荧光定量PCR和Western blot鉴定有效的干扰载体。选取最合适的干扰片段继续实验,绿色荧光蛋白挑取单克隆,荧光定量PCR、Western blot及免疫细胞荧光共聚焦等鉴定干扰后Tiam1基因／蛋白在M6细胞中的表达；利用MTT法、平板克隆、流式细胞周期检测Tiam1沉默后对M6细胞体外增殖能力的影响；细胞划痕和体外侵袭实验检测Tiam1对肝癌细胞迁移能力和侵袭能力的影响；裸鼠皮下瘤模型观察Tiam1对肝癌细胞体内成瘤增殖能力的影响；利用裸鼠尾静脉转移瘤模型观察Tiam1对肝癌细胞体内转移能力的影响。
4.统计学处理
用SPSS 16.0软件包进行数据分析,采用重复测量方差分析对比转染前后和干扰前后肝癌细胞株的MTT体外增殖实验、细胞划痕实验、皮下成瘤实验是否具有显著性差异；采用单因素方差分析对比转染前后和干扰前后肝癌细胞株的荧光定量PCR、平板克隆形成实验、流式细胞术细胞周期实验、Transwell侵袭实验是否具有显著性差异；多重比较采用LSD法；P<0.05为有统计学意义。
结果
1.Tiam1基因在肝癌细胞株和正常细胞株中的表达情况
采用荧光定量PCR法检测2种人肝癌细胞株和1种人正常肝细胞株中Tiam1基因的表达情况。单因素方差分析结果表明：与正常肝细胞株HL-7702相比,Tiam1基因在M6细胞高表达,差异具有显著性(P=0.000),在97L细胞低表达,差异无显著性(P=0.766);Western blot.免疫细胞化学、免疫细胞荧光、免疫细胞荧光共聚焦结果均表明Tiam1在HL-7702和97L中低表达,在M6中高表达。
2.稳定特异性表达Tiam1基因对肝癌细胞生物学特性的影响
成功构建了以绿色荧光蛋白为报告基因的Tiam1过表达载体pCDF1-Tiam1+-copGFP,进行慢病毒的包装和滴度测定后将该载体转染至低转移潜能的肝癌细胞株97L中,并设立空白对照,绿色荧光为标记物挑取3个阳性单克隆,荧光定量PCR.Western blot结果发现单克隆97L／T2转染效率最高；将其命名为97L/copGFP/Tiam 1+,同时空白对照细胞株命名为97L/copGFP/mock,未经任何处理的细胞为97L。
MTT法观察稳定表达Tiam1基因后体外细胞的增殖情况,不同细胞在不同时间增殖速度有显著性差异,并呈时间依赖关系(F=24.658,P=0.000)；平板克隆形成实验结果也显示97L/copGFP/Tiam 1+细胞的活力明显较其他两组强(P=0.028,P=0.044),流式细胞术结果显示97L/copGFP/Tiam 1+细胞中S期比例明显较其他两组增加(P=0.000,P=0.000),这些结果均说明Tiam1表达水平增加后,显著增强肿瘤细胞体外生长能力。
细胞划痕实验和体外侵袭小室检测稳定表达特异性Tiam1基因后细胞迁移和侵袭能力的改变,结果显示不同细胞在不同时间迁移细胞数有显著性差异,并呈时间依赖关系(F=93.331,P=0.000)；与97L/copGFP/mock细胞和97L细胞相比,97L/copGFP/Tiam1+细胞的侵袭能力明显增加,差异具有统计学意义(P=0.000,P=0.000)。这些结果表明Tiam1表达水平增加显著增强肝癌细胞迁移和侵袭能力。
通过肝癌可视化动物模型,我们观察稳定表达Tiam1基因后对肝癌体内增殖和转移能力的影响。将97L/copGFP/mock细胞和97L/copGFP/Tiam1+细胞接种于裸鼠双侧背部皮下,通过30天连续的观察,我们发现特异性表达Tiam1基因后,显著地增加了肝癌细胞的体内增殖能力(F=192.294,P=0.000),增殖差异从第15天开始,到第30天,差异达到最大,为2.1倍。采用尾静脉注射法建立了稳定表达Tiam1前后肝癌细胞的裸鼠体内静脉转移瘤模型。2个月后处死裸鼠并在整体荧光体视镜下观察发现在97L/copGFP/mock组的6只裸鼠中仅有1只可观察到微小肺转移；而97L/copGFP/Tiam1+组的6只裸鼠中有3只出现了肺转移,3只出现肝转移,2只出现骨转移；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肝癌细胞的转移。
3. Tiam1基因表达沉默对肝癌细胞生物学特性的影响
利用RNA干扰技术,成功构建了4个以绿色荧光蛋白为报告基因的Tiam1特异性的慢病毒干扰载体pGCSIL-EGFP,通过慢病毒介导的RNAi技术沉默具有高转移潜能的肝癌细胞株M6内源性Tiam1基因的表达,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(84.0%)利用含干扰片段B的慢病毒和空载体病毒感染M6细胞,绿色荧光挑选单克隆,荧光定量PCR和Western blot结果发现Tiam 1在干扰克隆M6／T2中表达最弱,干扰效率达(90.4%),以干扰克隆M6／T2作为Tiam1基因稳定沉默的肝癌细胞株,将其命名为为M6/EGFP/Tiam1-,同时空白对照细胞株命名为M6/EGFP/mock,未经任何处理的原始细胞为M6。
MTT法观察稳定表达Tiam1基因后体外细胞的增殖情况,不同细胞在不同时间增殖速度有显著性差异,并呈时间依赖关系(F=59.732,P=0.000)；平板克隆形成实验结果也显示M6/EGFP/Tiam1细胞的活力明显较其他两组减弱(P=0.000,P=0.000)；流式细胞术结果显示M6/EGFP/Tiam1细胞中S期比例明显较其他两组减小(P=0.000,P=0.000),这些结果均说明Tiam1表达水平减弱后,显著抑制了肿瘤细胞体外增殖能力。
细胞划痕实验和体外侵袭小室检测稳定沉默Tiam1基因后细胞迁移和侵袭能力的改变,结果显示不同细胞在不同时间迁移细胞数有显著性差异,并呈时间依赖关系(F=248.998,P=0.000)；与M6细胞和M6/EGFP/mock细胞相比,M6/EGFP/Tiam1细胞的侵袭能力明显减弱,差异具有统计学意义(P=0.000,P=0.000)。这些结果表明Tiam1表达水平降低显著抑制肝癌细胞迁移和侵袭能力。
通过肝癌可视化动物模型,我们观察稳定沉默Tiam1基因表达后对肝癌增殖和转移能力的影响。将M6/EGFP/mock细胞和M6/EGFP/Tiam1-细胞接种于裸鼠双侧背部皮下,通过30天连续的观察,我们发现特异性沉默Tiam1基因表达后,显著抑制了肝癌细胞的体内增殖能力(F=192.294,P=0.000),增殖差异从第15天开始,到第30天,差异达到最大,为2.2倍。采用尾静脉注射法建立了稳定沉默Tiam1表达前后肝癌细胞的裸鼠体内转移模型。2个月后处死裸鼠并在整体荧光体视镜下观察发现在M6/EGFP/mock组的6只裸鼠中有6只出现了肺转移,5只出现肝转移,6只出现骨转移；而M6/EGFP/Tiam1组的6只裸鼠中有1只可观察到肺转移,1只出现骨转移,未见肉眼可见肝转移；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肝癌的转移。
结论
1.利用慢病毒介导的稳定转染技术初步研究Tiam1基因在肝癌增殖、侵袭、转移中的作用,发现Tiam1基因是肝癌增殖、侵袭、转移的促进基因。
2.利用慢病毒介导的基因稳定沉默技术进一步验证Tiam1基因在肝癌增殖、侵袭、转移中的作用,从反面确证Tiam1基因是肝癌增殖、侵袭、转移的促进基因。
本研究的创新之处
1.本项目以慢病毒为载体,分别建立了稳定表达外源性Tiam1基因肝癌细胞株97L/copGFP/Tiam 1+和特异性Tiam1基因稳定沉默的肝癌细胞株M6/EGFP/Tiam 1-,为深入研究Tiam1基因的功能提供了有价值的工具。
2.通过对比观察表达Tiam1基因肝癌细胞株97L/copGFP/Tiam 1+和特异性Tiam1基因表达沉默肝癌细胞株M6/EGFP/Tiam1-细胞的体内、外生物学能力的改变,初步证实Tiam1基因与肝癌侵袭转移密切相关。
BACKGROUND & OBJECTIVE
Hepatocellular carcinoma (HCC) is one of the common cancers in the world. The incidence rate of HCC 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.
Tiam1(T lymphoma invasion and metastasis 1)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. 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 Tiaml 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.
The role of Tiaml 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 Tiaml in the metastasis of colorectal cancer is unclear.
In our previous study, by using tissue chip of normal tissues and common malignant tumors which was designed and produced by ourselves and immunohistochemistry, Tiaml protein expressions were detected in human normal tissues and common malignant tumors,213 cases of hepatocellular carcinoma tissues, 9 human hepatocellular carcinoma cell lines and 1 human normal hepatic cell. Tiaml protein was highly expressed in many tumors and it can be acted as one important tumor maker for tumorigenesis and development. Tiaml protein was up-regulated in hepatocellular carcinoma tissues, expressed lowly in liver sclerotic tissues and had no expression in normal hepatic tissue, indicating that Tiaml may be regarded as the important marker for clinical detection of HCC.Combined evaluation of Tiaml protein high expression and clinical follow-up data analysis showed that Tiam1 had close relationship with prognosis and metastasis of hepatocellular carcinoma patients.Tiam1 may act as the important marker for judging prognosis of HCC.
In this study, we aim to clarify the possible role of Tiaml gene in the proliferation, invasion and metastasis of HCC.It will be helpful to understand the molecular basis of HCC,and establish Tiaml as a new target for early metastatic diagnostic markers and novel therapeutic strategies.
METHODS
1.Expression of Tiaml gene/protein in human normal liver cells and liver cancer cells.
Real-time PCR, Western blot, Immunocytochemistry, Cellular immunofluoresce-nce and Cellular immunofluorescence confocal were used to examine the expressions of Tiaml in 2 hepatocellular carcinoma cell lines named M6,97L and 1 normal named HL-7702.
2.Effect of Tiaml overexpression on the biological behaviors of human HCC
Lentiviral vector pCDF1-Tiam1-copGFP was constructed by gene cloning. Recombinant lentivirus was harvested from 293FT cells cotransfected with the pPACKF1 Packaging Plasmid Mix.Tiam1/C1199HA cDNA was transfected into the 97L cell,which had lower Tiaml expression. Single transfectant cells clone were established 97L/copGFP/Tiaml+line by copGFP, Single mock cells clone were also established 97L/copGFP/mock line by copGFP. The Tiaml expression in transfectant was determined by RT-PCR, immunohistochemistry and Western blot. The biological behaviors of tranfectant were investigated by MTT assay, plate colony formation assay, flow cytometry, cell wounding heal assay, invasion assays in vitro, subcutaneous tumor model in nude mice, metastasis assay in vivo through the lateral tail-vein injection through LT-9MACIMSYSPULS system which were used to assess the functional effects of Tiaml silencing 97L cells in vitro or in vivo respectively.
3.Lentivirus-mediated silencing of Tiaml gene in human HCC M6 cell line.
Lentiviral expression vectors containing enhanced green fluorescence protein (EGFP) and Tiaml small interfering RNA (Lenti-Tiamlsi), or the control siRNA (Lenti-NC) gene were constructed. Human HCC M6 cell line was transfected with a different multiplicity of infection (MOI) of Lenti-Tiaml-si-A, B,C,D or Lenti-NC, and cultured to obtain stably-transfected M6/EGFP/Tiam1- and M6/EGFP/mock cells. The expression of Tiam1 mRNA/protein was determined by real-time PCR, western blot and confocal immunofluorescence.The biological behaviors of tranfectant were investigated by MTT assay, plate colony formation assay, flow cytometry, cell wounding heal assay, invasion assays in vitro, subcutaneous tumor model in nude mice, metastasis assay in vivo through the lateral tail-vein injection through LT-9MACIMSYSPULS system which were used to assess the functional effects of Tiam1 silencing M6 cells in vitro or in vivo, respectively.
4.Statistical analysis
Statistical analysis was done using SPSS 16.0. MTT assay, cell wounding heal assay, subcutaneous tumor model in nude mice were analyzed by repeated measure square analyze.Real-time PCR, plate colony formation assay, flow cytometry, invasion assays in vitro were analyzed one-factor analysis of variance.
RESULTS
The main results and findings are as follows:
1.The expressions of Tiaml gene in human normal liver cell line and liver cancer cell lines.
Tiam1 gene expressions in 2 human hepatocellular carcinoma cell lines and 1 human normal hepatic cell line were detected by real time PCR. The results of one-factor analysis of variance showed that the expressions of Tiam1 in 3 cell lines had significant difference (F=155.975,P=0.000).The expresions of Tiaml in M6 was higher than 97L and normal hepatic cell line HL-7702 with significant difference.
2.Effect of Tiam1 overexpression on the biological behaviors of human HCC
Recombinant pCDFl-Tiaml-copGFP lentivirus vector was constructed and identified by restriction endonuclease analysis and DNA sequencing.6771bp vector segment and 3800bp Tiam1 were got after EcoRI digested.After 60 hours of transferring pCDF1-Tiam1-copGFP and packaging plasmid into 293FT cell,the green fluorescence could be observed in some of the transferring cells.
97L cells were infected with recombinant lentivirus or mock lentivirus. A higher level of Tiaml expression was seen in the Tiaml transfected clone 2, we chose 97L/T2 clone (named 97L/copGFP/Tiam1+)cells and mock clone (named 97L/copGFP/mock) as function experiments in vitro or vivo.The result showed a significantly enhanced proliferation features compared with the 97L and 97L/copGFP/mock cells as determined by Real-time PCR, Western blot, Immunofluorescence confocal. The results showed that Tiaml overexpression promoted proliferation:MTT assay (F=24.658,P=0.000); plate colony formation assay(P=0.028,P=0.044); flow cytometry (P=0.000,P=0.000);cell wounding heal assay(F=93.331,P=0.000) and invasive capabilities(P=0.000,P=0.000)in 97L/copGFP/Tiaml+cells compared with 97L or 97L/copGFP/mock in vitro. Next, the effect of Tiaml on tumor growth was assessed by subcutaneous injection of 97L/copGFP/Tiam1+ and 97L/copGFP/mock cells for thirty days in vivo. Compared with 97L/copGFP/mock cells, the expression of Tiaml led to a pronounced increase in 97L/copGFP/Tiaml+cell growth starting from day 15,up to 2.1-fold increase in mice of tumor volume at day 30 after cell injection(F=192.294,P=0.000).To unambiguously elucidate the enhanced effect of Tiaml on HCC metastasis, we performed and metastasis assay in vivo through the lateral tail-vein injection. Mice were sacrificed 2 months later because the mice of 97L/copGFP/Tiaml+ group were moribund. The metastasis in the liver, lung, bone and other organs were determined the metastatic ability by whole-body visualizing orthotopic animal model and histological examination;in 97L/copGFP/Tiam1+ group,50%(3/6) of mice developed lung metastasis that appeared as numerous green nodules on the lung, 50%(3/6) of mice developed liver metastasis,34%(2/6) developed bone metastasis. In 97L/copGFP/mock group, only 29%(1/6) of animal had lung metastasis.
These data indicated that Tiaml expression in liver 97L cell was associated with enhanced migratory and invasive ability.
3.Effect of Tiaml silencing on the biological behaviors of human HCC M6 cells.To require prolonged suppression of Tiaml protein in M6 cells, we constructed 4 vshRNA (pGC-LV recombination vector) containing Tiam1 interfere sequens A, B, C and D.Real-time PCR analysis showed that the mRNA levels of Tiaml in four Tiaml-si cells were all suppressed, especially in Tiaml-si-B cells (84.0%).Then, we reinfected M6 cells.with si-B for further identifycation.
M6 cells were infected with recombinant lentivirus or mock lentivirus. A lower level of Tiaml expression was seen in the Tiaml transfected clone 2(90.4%), we chose M6/T2 clone (named M6/EGFP/Tiaml-)cells and mock clone (named M6/EGFP/mock) as function experiments in vitro or vivo.The result showed a significantly declined proliferation features of M6/EGFP/Tiam1- compared with the M6 and M6/EGFP/mock cells as determined by Real-time PCR, Western blot, Immunofluorescence confocal. The results showed that Tiam1 overexpression promoted proliferation:MTT assay (F=59.732,P=0.000);plate colony formation assay(P=0.028, P=0.044); flow cytometry (P=0.000, P=0.000); cell wounding heal assay(F=248.998,P=0.000) and invasion assays in vitro (P=0.000, P=0.000) in M6/EGFP/Tiam1- cells compared with M6 or M6/EGFP/mock in vitro.Next, the effect of Tiaml on tumor growth was assessed by subcutaneous injection of M6/EGFP/mock and M6/EGFP/Tiam1- cells for thirty days in vivo.Compared with M6/EGFP/mock cells, the silencing of Tiaml led to a pronounced decrease in M6/EGFP/Tiam1- cell growth starting from day 15,up to 2.2-fold decrease in mice of tumor volume at day 30 after cell injection(F=192.294,P=0.000). To unambiguously elucidate the declined effect of Tiaml on HCC metastasis, we performed and metastasis assay in vivo through the lateral tail-vein injection.Mice were sacrificed 2 months later because the mice of M6/EGFP/Tiam1- group were moribund.The metastasis in the liver, lung, bone and other organs were determined by the metastatic ability evaluated by whole-body visualizing orthotopic animal model and histological examination. In M6/EGFP/Tiam1- group, only 17%(1/6) of animal had lung metastasis,17%(1/6) had bone metastasis. But in M6/EGFP/mock group,100%(6/6) of mice developed lung metastasis that appeared as numerous green nodules on the lung,83%(5/6) of mice developed liver metastasis,100%(6/6) developed bone metastasis.
These data indicated that Tiam1 silencing in liver M6 cell was associated with decreasing migratory and invasive ability.
CONCLUSION
Tiaml gene plays an important role in proliferation, invasion and metastasis of HCC and is a metastasis-related gene.Clinically it may be a useful indicator of the tumor progression and metastasis in HCC.
引文
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