【作者】 郭磊；

【导师】 杨连粤； 袁志民；

【作者基本信息】 中南大学 ， 外科学， 2009， 博士

【摘要】 肝细胞癌(Hepatocellular Carcinoma, HCC)是世界上最常见、高发的恶性肿瘤之一,因其高发病率、高复发转移率及高死亡率,已成为一种威胁人民生命健康的最常见的恶性肿瘤。显然,深入研究HCC复发转移的分子机制并积极探索有效的抗复发转移的治疗措施,对于进一步改善HCC患者长期存活率具有重要意义。液泡膜蛋白1(Vacuole membrane protein 1,Vmp1)是新近发现的一类跨膜蛋白,在进化中高度保守,提示其可能参与蛋白间的相互作用和跨物种的重要生命活动。近年来的研究发现Vmp1为蛋白分泌、细胞器官形成及多细胞发育过程所必需,在诸多肿瘤相关的细胞学过程(包括细胞膜运输、生长增殖、自噬等)中发挥着关键的作用,提示Vmp1可能在肿瘤发生、发展中发挥了重要作用。更重要的是,Vmp1是细胞-细胞间连接及紧密连接形成的重要成分,其在调控细胞粘附能力方面的强大功能,提示Vmp1可能参与HCC侵袭转移过程,但其作用机制目前尚不清楚。因此,本课题在研究Vmp1在临床肝癌病例及肝癌细胞系中表达情况的基础上,进一步通过质粒构建转染技术上调Vmp1的表达,并综合利用一系列体外、体内方法研究Vmp1调控HCC侵袭转移的分子机制,同时探讨上调Vmp1表达对HCC侵袭转移的影响。我们获得了如下研究结果：1、我们采用半定量RT-PCR、荧光实时定量PCR和Western blot等方法检测了Vmp1 mRNA和蛋白在38例新鲜HCC组织及相应癌旁肝组织(PCLT)、5例门静脉癌栓(PVTT)中的表达水平,结果发现Vmp1 mRNA和蛋白在HCC组织中均明显低表达,在PVTT中的表达水平最低。免疫组化法检测124例HCC组织中Vmp1的表达,结果显示Vmp1主要分布于细胞浆内及胞膜,HCC组织中Vmp1蛋白阳性表达率(81／124,65.3%)明显低于PCLT组织(108／124,87.1%),且其表达水平与HCC的肿瘤结节数目、有无静脉浸润及有无包膜形成等临床病理特征密切相关。同时,结节性肝癌(NHCC)中的Vmp1蛋白表达水平明显低于孤立性大肝癌(SLHCC)和小肝癌(SHCC),而在SLHCC和SHCC间其表达无明显差异。Vmp1低表达组HCC患者的无瘤生存时间及总体生存时间均明显短于Vmp1高表达组的患者,多元Cox回归模型显示Vmp1低表达是HCC预后的独立危险因素,提示Vmp1的低表达与HCC的侵袭转移密切相关,并可能因此而导致了HCC患者的不良预后。2、RT-PCR及Western blot方法检测Vmp1 mRNA和蛋白在HepG2、MHCC97-L和HCCLM3这三种侵袭转移潜能依次升高的肝癌细胞系中的表达水平,并以常氏肝细胞系CCL13作为对照。结果显示Vmp1 mRNA和蛋白在HCCLM3和MHCC97-L细胞系中的表达明显低于HepG2和CCL13细胞系,且其表达水平在HepG2、MHCC97-L、HCCLM3细胞中依次降低,提示Vmp1的低表达与肝癌细胞的侵袭转移潜能密切相关。3、我们构建了重组质粒pCMVtag2C-VMP1,使用FuGENE 6转染并经G418筛选,获得了稳定转染细胞系HCCLM3VMP1+和空质粒对照组HCCLM3vector细胞。RT-PCR和Western blot结果发现HCCLM3VMP1+细胞中Vmp1的基因和蛋白的表达被明显上调。MTT法结果显示HCCLM3VMP1+较HCCLM3vector细胞的增殖能力明显下降；划痕实验显示HCCLM3VMP1+较HCCLM3vector细胞迁移能力明显下降,Transwell侵袭实验显示HCCLM3VMP1+细胞较HCCLM3vector细胞的侵袭能力明显下降；粘附实验结果显示与HCCLM3vector细胞相比,HCCLM3VMP1+细胞的同质性粘附能力明显增强而异质性粘附能力明显减弱。这些结果提示在体外实验中上调Vmp1的表达可显著影响肝癌细胞的粘附能力而抑制其增殖、迁移、侵袭。4、为在体内观察上调Vmp1对肝癌侵袭转移的影响,我们建立了HCCLM3转移性人肝癌细胞裸鼠模型。免疫组化结果显示,HCCLM3VMP1+组种植瘤组织中Vmp1的表达水平明显高于HCCLM3vector组；测量皮下种植瘤的大小,结果显示HCCLM3VMP1+组裸鼠原位种植瘤的平均体积明显小于HCCLM3vector组；裸鼠肺组织连续切片计数肺转移的数目,结果显示体内上调Vmp1能够抑制HCCLM3细胞的肺转移能力。我们的结果提示,上调Vmp1后在体内肝癌细胞的成瘤能力和侵袭转移能力受到明显抑制。5、已有研究显示Vmp1为细胞间连接和紧密连接形成的必要成分,在相邻细胞间的斑样结构与ZO-1共同定位。我们因而推测,Vmp1是否通过对ZO-1的调节来实现其对细胞粘附能力的影响。因此,我们用免疫共沉淀法证明HCCLM3VMP1+细胞中Vmp1与ZO-1存在直接的相互作用,但Western blot显示HCCLM3VMP1+和HCCLM3vector这两种细胞中ZO-1蛋白的表达水平并无明显差异。于是我们进一步研究Vmp1对ZO-1酪氨酸磷酸化的调控,免疫沉淀及Western blot的结果显示,HCCLMSP1+细胞中ZO-1蛋白的酪氨酸磷酸化水平明显低于HCCLM3vector。这些结果提示,上调Vmp1的表达可以降低ZO-1蛋白的酪氨酸磷酸化水平,从而上调ZO-1的功能以抑制肝癌的转移。基于上述研究结果我们可以得出以下结论：Vmp1在肝癌组织中低表达,其表达水平与肿瘤结节数目、包膜形成及静脉侵犯等临床病理特征密切相关,并显著影响肝癌患者的预后。研究证明,体内外上调Vmp1的表达可显著抑制肝癌的侵袭转移。Vmp1通过调节ZO-1的酪氨酸磷酸化调控肝癌细胞的粘附能力,从而在肝癌侵袭转移中发挥重要作用。本研究提示,Vmp1可作为一个新的肝癌预后标志物和潜在的侵袭转移干预靶点。更多还原

【Abstract】 Hepatocellular carcinoma (HCC)is one of the most common malignancies in the world.Because of the high incidence, high recurrence and high mortality, HCC has already become the important risk for the people’s health in China and all over the world. Thus,exploring further inhibition of invasion and metastasis is of great importance in the HCC therapies.Vacuole membrane protein 1 (Vmp1)is a recently identified conserved putative membrane protein, whose function is now beginning to be elucidated. Recent studies have demonstrated that Vmp1 is required for protein secretion, organelle organization, as well as organelle biogenesis and multicellular development. Vmp1 is recently identified to be importantly involved in cancer-relevant processes, including membrane traffic, proliferation, growth and autophagy. These results suggest a critical role of Vmp1 in tumor relevant cellular processes.More importantly and interestingly, recent reports showed that Vmp1 is an essential component of initial cell-cell contacts and tight junction formation, which playing a critical role in cell-cell adhesion. However, the function of this protein and its mode of action in tumor progression are still unknown. Therefore, we carried out the present study to determine the expression of Vmp1 in human HCC tissues as well as cell lines and attempted to elucidate the function of Vmp1 in the metastasis of HCC by characterizing its role in cell adherence via using both in vitro and in vivo models.1.Semi-quantitative PCR, real-time quantitative PCR and Western blot were employed to detect Vmpl mRNA and protein in 38 cases of HCC fresh tissues,paracarcinomatous liver tissues (PCLTs), and 5 portal vein tumor thromboses (PVTTs). Our results showed that both mRNA and protein levels of Vmpl were significantly decreased in HCC tissues than those in the corresponding PCLTs and NL tissues, even lower in PVTT compared to HCC.Immunohistochemical staining showed a cytoplasmic and membranic distribution of Vmpl,and the positive expression rate of Vmpl was significantly lower in HCC (81 of 124, 65.3%)than that in PCLT(108 of 124,87.1%). The Vmp1 expression levels were found significantly lower in HCCs with multiple nodules, without capsule formation and with vein invasion. Furthermore, the expression level of Vmpl protein was significantly down-regulated in nodular HCC (NHCC)when compared with solitary large HCC (SLHCC) and small HCC(SHCC).Moreover, HCC patients of the low Vmp1 expression group had both poorer disease free survival and poorer overall survival than those of the high Vmp1 expression group.By multivariable Cox regression analysis,low Vmp1 expression was found to be an independent prognostic factor for overall survival.2.We further confirm the Vmp1 expression in three HCC cell lines: HepG2, MHCC97-L and HCCLM3,with a liver cell line CCL13 as a control.The results of RT-PCR and Western blot showed a significantly lower expression of the Vmp1 mRNA and protein in HCCLM3 and MHCC97-L cells than those in HepG2 and CCL13 cells.Among the three HCC cell lines analyzed, HCCLM3 cells have the lowest Vmp1 expression, followed by MHCC97-L and HepG2.Of note is that the reduced expression of Vmp1 were in agreement with the metastatic potential of these HCC cell lines, suggesting a potential role for Vmp1 in HCC metastasis.3.To further define the correlation of Vmp1 expression and HCC metastasis,we employed plasmid transfection approach to up-regulate the expression of Vmp1 in HCCLM3 cells.We successfully constructed recombinant plasmid pCMVtag2C-VMP1.After FuGENE 6 transfection and three-week selection in media supplied with G418,HCCLM3 cell lines, stably transfected with the VMP1-expressing vector or control vector, were obtained and named as HCCLM3VMP1+ and HCCLM3vector respectively. The expression of Vmp1 mRNA and protein were markedly increased in HCCLM3VMP1+ compared with HCCLM3vector, which showed a satisfactory transfection efficiency. By MTT method, an obvious decrease of proliferation was observed in HCCLM3VMP1+ cells compared with HCCLM3vector cell.The wound-healing assay showed that the closure of HCCLM3VMP1+was significantly slower than that of HCCLM3vector. The results of Transwell invasion assay showed the number of HCCLM3VMP1+ cells that passed through matrigel was only 34% as compared with HCCLM3vector cells.Considering the close relation of Vmp1 with cellular adhesion, the adhesion test confirmed HCCLM3VMP1+ cells had significantly higher homogeneity adhesion and lower heterogeneity adhesion ability than HCCLM3vector cells.Together, these results support a critical role for Vmp1 in the adherence and metastasis of HCCLM3 cell.4.We further examined the in vivo relevance of the potential role for Vmp1 in HCC tumorigenesis and metastasis by using a nude mice implantation model.Primary subcutaneous tumor of HCCLM3 was measured every five days and mice were sacrificed 35 days after implantation. We found that the average size of primary tumors in HCCLM3VMP1+ group was dramatically smaller than that of HCCLM3vector group,which suggested that up-regulation of Vmp1 in vivo could inhibit growth of HCC.The expression of Vmp1 in HCCLM3 tumor was determined to ensure the difference of Vmp1 expression in vivo.The results showed that the expression of Vmp1 was significantly increased in HCCLM3VMP1+ tumor than that in HCCLM3vector tumor. The pulmonary metastasis was observed in the lung tissue sections of only two mice in HCCLM3VMP1+ group (two of seven,28.6%),significantly less than the ratio of pulmonary metastasis in HCCLM3vector group(six of seven,85.7%).Together, these data support an important role for Vmp1 in HCC metastasis.5.Since Vmp1 is essential for cell-cell contacts and tight junction formation, where it colocalizes with ZO-1 in spots between neighboring cells, we further explore Vmp1’s regulating effect on ZO-1.Firstly, co-immunoprecipitations confirmed the direct interaction of Vmp1 and ZO-1 proteins in HCC cells.Next, the corresponding ZO-1 protein expression of HCCLM3VMP1+ and HCCLM3vector cells were detected, but revealed no significant change at total protein level.Further immunoprecipitation and Western blotting of HCCLM3 MP1+ and HCCLM3vector cells showed ZO-1 was highly tyrosine-phosphorylated in HCCLM3vector, but was dephosphorylated in HCCLM3VMP1+,confirming the hypothesis that up-regulation of Vmp1 decreases the tyrosine phosphorylation of ZO-1,which leads to up-regulation of the function of ZO-1,and these phenomena contribute to inhibition of HCC metastasis.In conclusion, our study has shown for the first time that down-regulation of Vmp1 significantly correlates with poor prognosis of HCC.Furthermore, we have demonstrated Vmpl inhibits metastasis of HCC by affecting cell adherence through reduction of ZO-1 tyrosine phosphorylation. Collectively, our data suggests Vmp1 as a novel prognostic marker and a potential therapeutic target for metastasis of HCC.更多还原