【作者】 汪淑晶；

【导师】 张嘉宁；

【作者基本信息】 大连医科大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 胞膜窖(caveolae)是50～100nm烧瓶状的细胞膜结构,和信号传导密切相关。窖蛋白-1(caveolin-1)是胞膜窖的主要结构成分,在细胞内吞、胆固醇运输、信号传导、肿瘤转化中发挥着重要的作用。Caveolin-1(Cav-1)在肿瘤中是发挥抑癌作用还是促癌作用一直存在争论。在大多数肿瘤细胞如乳腺癌、肺癌、卵巢癌中发挥抑癌基因样作用,而在少数肿瘤如前列腺癌中则发挥癌基因样作用。然而目前对Cav-1在肝癌细胞中所发挥作用的研究尚少,对于Cav-1介导肝癌细胞凋亡、侵袭和转移的分子机制亦不十分清楚。我们前期研究结果表明,Cav-1在小鼠肝癌细胞Hca-F和Hca-P中呈高表达,且前者高于后者,在Hepa1-6细胞中表达缺失。其中Hca-F具有高侵袭、高淋巴道转移潜能,Hca-P具有低侵袭、低淋巴道转移潜能,Hepa1-6无转移潜能。因此推测,Cav-1的表达可能在小鼠肝癌细胞恶性表型转化中发挥重要作用。大量的实验已经表明,肿瘤细胞的恶性行为很大程度上是由细胞表面形成过量的β1,6GlcNAc分支结构,进而产生多天线的N-糖链结构,从而改变了糖蛋白分子的生物学性状,使肿瘤细胞的生物学行为发生改变。然而,Cav-1是否影响小鼠肝癌细胞N-糖蛋白的β1, 6GlcNAc分支结构,有关报道较少。CD147,又称基质金属蛋白酶诱导物(EMMPRIN),是属于免疫球蛋白超家族的跨膜糖蛋白,由于N-糖基化程度的不同,表现为分子量不同的高糖型(HG-CD147)和低糖型(LG-CD147)。已有研究证实,CD147在许多恶性肿瘤细胞中有表达,并能诱导基质金属蛋白酶产生,从而影响肿瘤细胞的侵袭和转移。我们前期研究结果显示,HG-CD147和LG-CD147在小鼠肝癌细胞Hca-F,Hca-P和Hepa1-6中均有表达,且Hca-F细胞的表达强度明显高于Hca-P和Hepa1-6细胞。此外,Cav-1的表达有利于LG-CD147向HG-CD147的转化。然而,CD147在不同淋巴道转移潜能的小鼠肝癌细胞株中的糖链结构如何,Cav-1表达是否对CD147糖蛋白糖链的分支结构产生了影响,Cav-1如何调节CD147的糖基化,有关报道甚少。本文在利用分子克隆、基因转染及RNA干扰等技术上调和下调Cav-1表达的基础上,1)观察Cav-1表达变化对小鼠肝癌细胞转化、凋亡、侵袭和转移等生物学行为的影响;2)阐明Cav-1介导小鼠肝癌细胞凋亡、侵袭和转移的分子机制;3)分析CD147在小鼠肝癌细胞株中的糖型结构,并初步探讨Cav-1对N-糖蛋白分支结构的调节作用。本研究实验结果如下:1. pEGFP-N2/Cav-1表达载体构建及在小鼠肝癌Hepa1-6细胞中的稳定表达利用RT-PCR从小鼠肺组织总RNA中成功克隆出546bp的Cav-1cDNA的24-557bp片断,重组阳性克隆经酶切和测序鉴定证实目的基因已正确插入pEGFP-N2表达载体中,且与载体上的GFP基因处于同一开放阅读框,记为pEGFP-N2/Cav-1。利用脂质体转染技术,G418筛选获得2个稳定表达外源Cav-1的Hepa1-6细胞株,记为Hepa1-6/Cav-1。RT-PCR和Western-blot方法证实,外源Cav-1基因整合到Hepa1-6细胞基因组中。免疫细胞化学和电镜结果显示,Cav-1在Hepa1-6细胞胞膜和胞浆中均有表达。2. Cav-1表达上调或下调对小鼠肝癌细胞转化及凋亡的影响软琼脂克隆形成实验和体内致瘤实验结果显示,与转染空载体组细胞(Hepa1-6/mock)和未转染组细胞Hepa1-6比较,Hepa1-6/Cav-1组细胞集落形成数和体内成瘤能力明显增加(P < 0.05)。RT-PCR和Western-blot结果显示,与scrambled siRNA和unrelated siRNA(control-siRNA)相比,Cav-1特异性siRNA(Cav-1-siRNA)能够有效下调Cav-1在小鼠肝癌H22细胞中的表达,并可持续至转染后3周左右。致瘤实验和TUNEL实验结果显示,与对照组细胞相比,Cav-1-siRNA转染的H22细胞(H22/Cav-1-siRNA)在软琼脂中的克隆形成数明显减少,体内成瘤能力下降,凋亡数增多(P < 0.05)。3. Cav-1在放线菌素D诱导小鼠肝癌细胞凋亡中的作用及分子机制Hoechst 33342染色结果显示,放线菌素D可诱导小鼠肝癌细胞发生凋亡,呈时间和浓度依赖性。TUNEL结果显示,Cav-1过表达减少放线菌素D诱导的Hepa1-6细胞凋亡数目;Cav-1下调增加Hca-F细胞凋亡数目,并增强Hca-F细胞对放线菌素D的敏感性。Western-blot结果显示,Cav-1过表达上调survivin蛋白水平,降低活性caspase-3(active caspase-3)表达;Cav-1表达沉默降低survivin蛋白水平,升高active caspase-3表达。4. Cav-1对小鼠肝癌细胞N-聚糖β1,6GlcNAc分支的调节PHA-L Lectin-blot结果显示,当Cav-1表达沉默时,H22细胞总蛋白与PHA-L的结合信号减弱,β1,6GlcNAc分支形成受抑制;当Cav-1过表达时,Hepa1-6细胞总蛋白与PHA-L的结合信号增强,β1,6GlcNAc分支形成增加。基质胶侵袭实验和体内淋巴结转移实验结果显示,β1,6GlcNAc分支减少可抑制H22细胞的侵袭和转移能力,β1,6GlcNAc分支增多可增强Hepa1-6细胞的侵袭能力。糖苷酶酶切实验结果显示,在小鼠肝癌Hca-F和Hepa1-6细胞中CD147为N-糖蛋白,LG-CD147均为高甘露糖型,HG-CD147分别为杂合型和复合型。免疫共沉淀及Lectin-blot分析结果显示,Cav-1过表达可增加CD147的平分型GlcNAc分支形成,而对β1,6GlcNAc分支无影响。Real-time PCR结果显示,Hepa1-6/Cav-1细胞中N-乙酰氨基葡萄糖基转移酶Ⅲ(GnTⅢ)的mRNA表达水平较Hepa1-6中升高,GnTⅣ有所降低,GnTⅤ升高不显著。结论:1. Cav-1可正性调节小鼠肝癌细胞转化、生存、侵袭和转移等恶性表型,发挥癌基因样作用。2. Cav-1在放线菌素D诱导小鼠肝癌细胞凋亡中发挥重要作用,可通过调节survivin/caspase-3生存通路介导细胞凋亡。3. Cav-1可通过调节小鼠肝癌细胞N-聚糖β1,6GlcNAc分支的形成介导肿瘤细胞的侵袭和转移。更多还原

【Abstract】 Caveolin-1 is a major structural protein of caveolae and plays important roles in signal transduction, cellular transformation and tumor metastasis. Currently, the exact functions of Cav-1in tumor remain controversial. Cav-1may act as a tumor suppressor in most (but not all) cancer cells, and Cav-1also acts as a tumor promoter in certain tumor cell types. Thus, the Cav-1gene has been regarded as both a tumor suppressor gene and an oncogene. However, the exact role of Cav-1in hepatoma remains unknown. Therefore, an understanding of the molecular mechanisms involved in tumor cell transformation and progression could be helpful in developing more effective treatments for hepatoma.In previous study, we have reported that Cav-1was highly expressed in mouse hepatoma cells with lymphatic metastasis potential, and could increase their invasive ability by up-regulating CD147 glycosylation level. This suggested that Cav-1expression might be correlated with the malign- nant phenotypes of hepatoma cells. However, the role of Cav-1in cell transformation and apoptosis has not been reported. Here,the relationship between Cav-1expression and malignant phenotypes was studied through overexpression of Cav-1in Cav-1-negative mouse hepatoma Hepa1-6 cells and gene-specific suppression of Cav-1expression in Cav-1-positive Hca-F cells.In this study, exogenous expression of Cav-1in Hepa1-6 cells increased their transformation potential both in vitro and in vivo, and prevented ActD- induced apoptosis via the activation of survivin-mediated survival pathway. By contrast, down-regulation of Cav-1in Hca-F cells using small inter- fering RNA significantly attenuated cell transformation ability, and induced apoptosis and enhanced cell sensitivity to ActD. These results suggest that Cav-1could play an active role in mediating cell transformation and survival as well as cell invasion, and thus acts as a tumor promoter in hepatoma cells. Moreover, Cav-1might be a potential target for gene and drug treatment of hepatoma.In addition, to further explore the exact roles of Cav-1and the possible utility of Cav-1downregulation for hepatoma therapy, we used RNAi technique to knockdown the expression of Cav-1in H22 hepatoma cells. In the present study, we have examined the phenotypic changes, including transformation capability and apoptotic potential, resulting from the reduction of Cav-1expression in transfected H22 cells both in vitro and in vivo. The results showed that Cav-1-siRNA could effectively inhibit the expression of Cav-1in H22 cells, and attenuate the transformation and survival capability of H22 cells in vitro and in vivo. Thus, Cav-1gene can be regarded as a very good target gene in genetic therapy for hepatoma and the use of Cav-1-siRNA deserves further investigations as a novel approach to cancer therapy.CD147, also known as matrix metalloproteinase inducer (EMMPRIN), is a highly glycosylated immunoglobulin superfamily transmembrane pro- tein. As a result of heterogeneous N-glycosylation, CD147 exists in both highly glycosylated form, HG-CD147 (40–60 kDa) and lowly glycosylated form, LG-CD147 (32 kDa). CD147 was enriched on the surface of many malignant tumor cells, and can induce matrix metalloproteinase (MMP) production, thereby affect tumor cell invasion and metastasis.Many studies have shown that the malignant phenotypes of tumor cells are correlated with the exce- ssive formation of cell surfaceβ1, 6GlcNAc branching structure. However, the effect of Cav-1on N-glycoproteinβ1, 6GlcNAc branch structure of mouse hepatoma cells is rarely reported.Our previous study showed that the CD147 expression level in Hca-F cells was significantly higher than that in Hca-P and Hepa1-6 cells. Cav-1expression resulted in the transformation from LG-CD147 to HG-CD147. However, there are few reports on the sugar chain structure of CD147 in different lymphatic metastasis potential of mouse hepatoma cell lines. The mechanism of the effect of Cav-1 on the CD147 glycosylation remains undetermined. In this study, we found that Cav-1silencing could inhibitβ1, 6GlcNAc branching formation, and decrease the invasive and metastasis ability of mouse hepatoma H22 cells. Conversely, Cav-1overexpression could upregulateβ1, 6GlcNAc branching formation, and increase the migratory and invasive ability of mouse hepatoma cells.In conclusion, Cav-1plays an active role in mediating cell transformation and survival, and is therefore regarded as an oncogene in hepatoma cells. Cav-1is involved in the hepatoma cell apoptosis through regulation of survivin-mediated survival pathway. Cav-1is involved in the hepatoma cell invasion and metastasis through regulation of N-glycanβ1, 6GlcNAc branching formation. These findings may implicate, at least partially, that Cav-1could represent an appropriate target for drug treatment of hepatoma.更多还原