【作者】 张文；

【导师】 查锡良； 王丽影；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 肝癌的恶性程度高，发生发展迅速，预后不良。因此对其发生和发展的机制进行研究显得很有必要。过氧化物酶体增生物激活受体Y（peroxisome proliferatoractivated receptor，PPAR）及全反式视黄酸（atRA）相关的信号通路活化都后可以对包括肝癌在内的多种肿瘤发挥抑制效应，包括抑制细胞增殖、诱导细胞分化和凋亡、降低肿瘤侵袭能力等。但是，目前对这两条信号通路分子机制的了解并不完全清楚，对其进行研究可以为临床治疗肝癌提供新的思路和治疗手段。因此，我们拟从这两条信号通路着手探讨这两条信号通路对肿瘤细胞增殖和迁移的影响及相应的分子机制。我们的研究分为三个部分。第一部分PTEN参与PPARγ相关信号通路抑制人肝癌细胞迁移的分子机制使用PPARγ的特异性配体（rosiglitazone，ROS）处理肝癌细胞系BEL-7404细胞后，发现ROS可以明显抑制其迁移但对其增殖没有显著影响。同时，在ROS处理后，BEL-7404细胞内的粘着斑激酶（focal adhesion kinase，FAK）及蛋白激酶B（protein kinase B，PKB，Akt）的磷酸化水平显著降低，然而胞内的有丝分裂原活化激酶1／2（Mitogen-activated protein kinases，MAPK；extracellularsignal-regulated kinases，ERKs）的磷酸化水平却没有变化。由于FAK和Akt都是抑癌基因PTEN（phosphatase and tensin homolog deleted on chromosome ten）的下游信号分子，因此我们检测了ROS对PTEN的影响，结果发现ROS可以上调PTEN蛋白质，而且这种上调作用具有剂量和时间依赖性；当使用PPARγ的特异性阻断剂BADGE（Bisphenol A diglycidyl ether）预处理细胞后，ROS对PTEN的上调作用不再出现，提示ROS上调PTEN是通过激活PPAR7来实现的。ROS处理后，PTEN的mRNA水平也出现升高，然而其基因的转录活性却没有明显上调，说明虽然激活PPARγ能够上调PTEN的mRNA，但对蛋白的上调并非通过增加其基因转录来实现。在BEL-7404细胞内转染PTEN的表达质粒后能够明显抑制该细胞的迁移。而转染PTEN的RNA干扰质粒pPTENi阻断PPARγ激活所致的PTEN上调后，ROS对BEL-7404细胞迁移的抑制作用不再出现。这提示PTEN的上调是ROS抑制细胞迁移所必须的。综上所述，该部分实验提示：ROS与PPARγ结合后可以通过上调PTEN蛋白质水平来抑制BEL-7404细胞的迁移能力。第二部分关于人肝癌细胞中PTEN负反馈调控的研究为进一步探讨PTEN的调控机制，我们将GFP-PTEN表达质粒转染人肝癌细胞系SMMC-7721，发现较高水平的PTEN总蛋白水平导致内源性PTEN蛋白质的水平下降，提示SMMC-7721细胞内PTEN存在负反馈调节机制。同时还观察到：GFP-PTEN表达质粒转染后内源性PTEN的基因转录活性和mRNA水平均出现明显下调，说明PTEN的负反馈调节是通过抑制基因的转录降低mRNA水平来实现的。使用Akt的持续活化质粒myr-Akt与GFP-PTEN表达质粒共转染SMMC-7721细胞后，尽管总的PTEN蛋白质水平仍然很高，但内源性PTEN未再出现下调；使用PI3K的阻断剂LY294002处理SMMC-7721细胞导致PTEN蛋白质和mRNA水平下调，说明PTEN的负反馈调节是通过PTEN抑制P13K／Akt通路来实现的。另外，LY294002处理Changs细胞和ES-2细胞后，细胞内的PTEN也出现下调，提示对PI₃K／Akt通路的抑制下调PTEN并非SMMC-7721细胞所独有。使用构建的PTEN片断突变质粒转染细胞后，发现C端缺失突变的质粒不能导致内源PTEN的下调，而N端缺失突变质粒可以诱导内源PTEN下调，提示PTEN的C端结构域是实现负反馈调控所必需的。综上所述，该部分实验提示：PTEN上调后抑制PI3K／Akt途径来下调自身的转录，从而实现负反馈调控。第三部分Profilin 1参与全反式视黄酸抑制人肝癌细胞的增殖和迁移的分子机制研究本实验室的前期研究发现，atRA诱导人肝癌细胞SMMC-7721可导致细胞内的profilin 1（PFN1）上调。使用westernblot对4例肝癌和癌旁组织中的PFN1进行分析发现，肝癌组织中的PFNl水平低于癌旁组织；对包含30例肝癌和癌旁组织的组织芯片进行PFN1的免疫组织化学染色得到同样的结果，提示PFN1的低表达可能与肝癌的恶性行为有关。为进一步的探讨PFN1在atRA抑制细胞增殖和迁移效应中的作用，我们构建并筛选了有效的针对PFN1的RNA干扰质粒。实验发现，使用PFN1的RNA干扰质粒转染阻断atRA诱导的PFN1上调后，atRA对SMMC-7721细胞增殖和迁移的抑制明显减弱。这提示PFN1的上调作用是atRA抑制SMMC-7721细胞增殖和迁移所必须的。更多还原

【Abstract】 Hepatocelluar Carcinoma (HCC) is a common malignant tumor in China. Metastasis and recurrence are the major causes for its bad prognosis. Because of the limitations in conventional therapy methods, it is necessary to make research into the molecular mechanisms of HCC development and migration. In many types of cancers, the activation of signaling pathways associated with PPARγ (peroxisome proliferator activated receptor γ) and atRA (all-trans retinoic acid) has been shown to have widely antitumor effects, such as inhibiting proliferation and invasion, inducing differentiation and apoptosis, et al. Although such effects have been reported in many studies, the potential mechanisms still remain to be further investigated. Therefore further study on molecular mechanisms of these pathways will supply new insights and methods to HCC therapy. The purpose of our study is to elucidate the effect of these pathways on proliferation and migration of hepatocarcinoma cells.Part 1Up-regulation of PTEN was involved in inhibition of migration by activation of PPARγ signaling in human hepatocelluar carcinoma cellsBEL-7404 cell was shown to have lower ability of migration after it was treated with rosiglitazone (a selective ligand of PPAR) as well as rosiglitazone treatment did not interfere in its proliferation. The phosphorylation of Akt and FAK were significantly attenuated in cells treated with rosiglitazone. However, the phosphorylation of ERK did not significantly change. Because both Akt and FAK are on the downstream of tumor suppressor PTEN, we detected the expression of PTEN in cells treated with rosiglitazone and found rosiglitazone could increase PTEN protein expression in a time- and dose- dependent manner, which depended on the activation of PPARγ. Rosiglitazone treatment also increased PTEN mRNA level. Furthermore, PTEN overexpression resulted in the inhibition of cell migration and PTEN knock-down blocked the effect of rosiglitazone on cell migration. It suggested that PTEN was required for rosiglitazone-induced migration inhibition in BEL-7404 cells. In conclusion, our results demonstrated that PTEN played a critical role in rosiglitazone-induced migration inhibition in BEL-7404 cell.Part 2 Mechanism of PTEN negative feedback regulation in human hepatocarcinomacell line SMMC-7721To further investigate mechanism of PTEN regulation, GFP-PTEN expression plasmids was introduced into SMMC-7721 cell (human hepatocarcinoma cell line). We observed that higher amount of PTEN led to lower endogenic PTEN both in protein level and mRNA level. These results implied there might be a feedback regulation of PTEN in SMMC-7721 cell. Furthermore, activated Akt blocked the inhibition effect of ectopic PTEN on endogenic PTEN and PI3K inhibitor LY294002 treatment resulted in decreasing PTEN both in protein and mRNA levels. These results suggested the feedback regulation of PTEN depended on inhibition of PDK/Akt pathway. LY294002-induced inhibition of PTEN was also found in ES-2 and Changs cells, suggesting the feedback regulation of PTEN might exist in other types of cell. Using fragment deletion plasmids of PTEN, we found that the inhibition effect of ectopic PTEN on endogenic PTEN depended on its Carboxyl-terminal region.Part 3Up-regulation of Profilin 1 was involved in atRA-induced inhibition of proliferation and migration in human hepatocelluar carcinoma cellsOur previous research showed that atRA could increase profilin 1 protein expression. Using western blot and immunohistochemical stain, we also found the protein level of profilin 1 was lower in hepatocarcinoma tissues than corresponding matched paracancerous tissues. Futhermore, we constructed SiRNA plasmid targeting PFN1 to perform further research. Using contracted SiRNA plasmid, we found RNAi-based PFN1 knockdown could rescue the inhibitory effect of ATRA on cell proliferation and migration. These results suggest that profilin 1 plays a role in the inhibitory effect of atRA on cell migration suppression in SMMC-7721.更多还原