摘要
第一部分p28~(GANK)增强肝癌细胞氧化应激耐受的作用与机制研究
研究背景和目的
肝细胞癌(Hepatocellular Carcinoma,HCC)是肝癌中最常见的类型,占所有原位肝癌病例的90%以上。HCC的发生发展是由体内多基因参与、多步骤协同的复杂过程,在其中氧化应激具有非常重要的作用。有综述总结了各种HCC动物模型的致癌机制:所有的HCC动物模型有唯一一个共同的特点,即肝细胞内活性氧自由基(reactive oxygen species, ROS)水平升高。细胞内ROS的水平决定于ROS的生成及清除能力,当ROS生成增多或者(和)细胞ROS清除能力减弱时,便会出现ROS水平升高。ROS化学性质活泼且具有氧化性质,能够导致DNA损伤、脂质氧化以及一些蛋白生物学功能的改变,造成细胞损伤,使细胞处于氧化应激状态。肝癌细胞的快速增殖导致大量ROS生成使细胞面临氧化应激,但是,已有研究证实相对于正常肝细胞,肝癌细胞具有更强的抗氧化酶体系和抗氧化能力,致使其在肝脏的过氧化环境中取得生存优势,这是各种因素导致肝癌发生的机制之一。另外,在肝癌的化疗过程中,大部分化疗药是通过直接或者间接诱导细胞中ROS水平升高诱导肝癌细胞死亡而发挥治疗作用的,但肝癌细胞强大的抗氧化系统能够保护肝癌细胞对抗这种死亡,而使肝癌的化疗效果不明显。综合已有研究成果,纵观肝癌的发生发展、转移、复发到肝癌的化疗耐受都与肝癌细胞对氧化应激的耐受有关。因此,寻找影响肝癌细胞氧化应激耐受力的关键分子具有重要意义。
gankyrin基因是2000年Fujita等人应用消减杂交法从人肝细胞肝癌组织中筛选出的一条新基因。它编码的Gankyrin蛋白在大部分肝癌组织(~70%)中呈高丰度表达,而且在肝癌生成的早期即已高表达,而在正常肝组织没有表达。本实验室和其他研究小组的研究已揭示p28~(GANK)蛋白的生物学功能是多样的:它既可以通过促进p53与其泛素连接酶MDM2结合而促进p53蛋白的泛素化及降解发挥抗凋亡作用,又能够作为穿梭蛋白将RelA转位到胞浆而抑制NF-κB转录活性,还可以增强Akt蛋白磷酸化而转导细胞生长信号。而p53蛋白、NF-κB转录因子和PI3K/AKT信号通路都在细胞的氧化应答中具有重要作用。我们实验室的研究结果还显示,利用腺病毒介导的方法在肝癌细胞系中干扰p28~(GANK)的表达能使细胞中ROS累积增多,导致自发性的细胞死亡;ROS清除剂NAC可以逆转这种细胞死亡的发生。这提示p28~(GANK)能够影响细胞的ROS累积,从而增强肝癌细胞对氧化应激的耐受力。
本研究拟利用H_2O_2这种经典的氧化应激诱导剂在肝癌细胞中诱导氧化应激,一方面观察氧化应激对肝细胞中p28~(GANK)表达水平的影响;另一方面观察改变肝癌细胞的p28~(GANK)蛋白表达水平后,细胞对氧化应激的耐受力的变化;并进一步探讨p28~(GANK)调控肝癌细胞氧化应激耐受的具体分子机制。
实验方法:
1.用不同浓度H_2O_2刺激细胞,利用real-time PCR、western blot等方法观察肝细胞(肝癌细胞)中p28~(GANK)基因转录以及翻译表达水平的变化;并用ROS清除剂NAC处理细胞,然后western blot检测细胞p28~(GANK)表达水平的变化;
2.利用GSH检测专用试剂盒分析DEN诱导SD大鼠肝癌模型过程中大鼠血浆GSH水平并通过real-time PCR的方法检测相应肝组织样本中p28~(GANK)基因表达变化情况;
3.通过质粒稳定转染、瞬时转染以及腺病毒感染的方法改变肝癌细胞中p28~(GANK)蛋白表达,然后通过观察显微镜下细胞形态变化、细胞PI染色阳性率以及PARP蛋白的剪切体等,观察p28~(GANK)高低表达对氧化应激诱导的细胞死亡的影响;
4.用ROS特异的荧光探针对H_2O_2刺激下的细胞或者转染了癌基因ras表达质粒的细胞进行染色,然后进行荧光显微镜镜下观察以及流式细胞仪检测,观察细胞内p28~(GANK)表达水平的变化对氧化应激下细胞内ROS累积的影响;
5.应用real-time PCR方法检测p28~(GANK)高低表达对氧化应激下抗氧化酶系统的诱导表达的影响以及肝细胞癌组织样本中p28~(GANK)表达与抗氧化酶系统表达水平的相关性;
6.用细胞总抗氧化能力试剂盒检测p28~(GANK)高低表达的稳定细胞系其抗氧化能力,观察这些细胞系总抗氧化能力是否不同;
7.电镜观察稳定细胞系HCCLM3H/CCLM3-sip的细胞内线粒体密度,并通过real-time PCR的方法检测两种细胞中线粒体生成相关基因的表达水平;
8.应用western blot方法检测p28~(GANK)高低表达对氧化应答关键信号通路:MAPK信号通路及PI3K/AKT信号通路的影响;并应用相应通路抑制剂观察以上通路在p28~(GANK)抗氧化功能中的重要性。
结果:
1.适度浓度的H_2O_2刺激能够增加肝癌细胞中p28~(GANK)转录及蛋白水平的表达,持续增加H_2O_2浓度则会使p28~(GANK)表达减少,视细胞类型不同具体刺激浓度亦不同;NAC处理能够降低SMMC7721细胞中p28~(GANK)表达水平;
2. DEN诱导大鼠肝癌过程中,大鼠血浆中GSH浓度变化与其肝组织中mRNA表达水平呈现负相关性;
3.增加(或者降低)细胞中p28~(GANK)蛋白的表达能够相应减少(或者增加)H_2O_2刺激诱导的细胞死亡,其中p53蛋白不发挥主要作用;
4. p28~(GANK)能够抑制H_2O_2刺激导致的ROS水平升高,并且能够降低ras表达质粒转染引起的细胞内ROS累积;
5. p28~(GANK)能够增强氧化应激下抗氧化酶的转录激活;
6. p28~(GANK)正向调节细胞的总抗氧化能力;
7.稳定干扰HCCLM3细胞内p28~(GANK)表达会降低细胞内线粒体密度,并且抑制线粒体生成相关基因的表达;
8. p28~(GANK)显著增强氧化应激导致的Akt的磷酸化,而且应用PI3K/AKT信号通路抑制剂可以减弱氧化应激下p28~(GANK)对细胞的保护作用。
结论:
本研究利用H_2O_2诱导氧化应激的模型研究了p28~(GANK)表达与细胞氧化应激耐受性间的关系。发现p28~(GANK)蛋白能够正向调节肝癌细胞对氧化应激的耐受力,而且一定浓度内的H_2O_2刺激能够上调肝癌细胞中p28~(GANK)的表达。氧化应激下p28~(GANK)能够增加肝癌细胞中抗氧化酶的表达,增强细胞的总抗氧化能力,减少细胞内ROS的累积,抑制氧化应激引起的细胞死亡。此外,氧化应激下p28~(GANK)还可以通过增强Akt蛋白磷酸化而保护肝癌细胞对抗氧化应激诱导的细胞死亡。
第二部分p28~(GANK)正反馈调节β-catenin信号通路的作用与机制研究
研究背景和目的
肝癌是世界第六大常发肿瘤,更因为其化疗敏感性低、高复发、预后差等原因被称为“癌中之王”。随着分子生物学的迅猛发展,以癌基因为靶点的生物学治疗被认为是未来治疗肝癌的有效手段。近年来一系列研究显示,肝癌癌蛋白p28~(GANK)有望成为肝细胞癌治疗的一个有效靶标。一方面,p28~(GANK)几乎表达于所有的肝细胞癌组织中,癌高于癌旁的几率约为百分之七十,但在正常肝组织中没有表达;另一方面p28~(GANK)能够促进细胞增殖,将正常细胞转化为癌性细胞。目前研究已经得知p28~(GANK)加快细胞周期进程,促进细胞增殖是通过增强抑癌蛋白Rb的磷酸化,加速Rb蛋白降解,促进转录因子E2F的释放而完成的。然而大部分肝癌细胞中都存在Rb的缺失或者突变,那么除Rb蛋白外,p28~(GANK)还能够通过哪些途径促进肝癌细胞的增殖呢?这个问题目前还没有研究涉及。
β-连环蛋白(β-catenin)是一种重要的肿瘤相关基因,除了在细胞粘附中有重要作用外,还是Wnt信号通路的重要功能分子。Wnt/β-catenin信号通路是众多组织中调控细胞增殖、凋亡和分化的重要通路。近来研究发现,在肝癌发生早期存在β-catenin蛋白的异常聚集,并且这种聚集能够促进肝癌的发生以及早期肝癌细胞的快速生长。探讨β-catenin在肝癌发生发展中的作用具有重要临床价值。
已有研究表明提高野生型p53水平可以明显下调β-catenin蛋白水平。而p28~(GANK)能与p53蛋白的泛素连接酶MDM2相互作用,促进p53蛋白的泛素化及其蛋白酶体途径依赖的蛋白降解,我们推测在肝细胞癌的发生发展中癌蛋白p28~(GANK)与β-catenin间可能存在相互联系。
本研究将利用一些经典的分子生物学实验方法研究:在各种细胞生长信号的刺激下p28~(GANK)表达水平的改变;改变p28~(GANK)蛋白表达对β-catenin信号的影响;介导生长因子刺激信号的关键蛋白Akt对p28~(GANK)表达的影响;Wnt/β-catenin信号通路激活对细胞中p28~(GANK)的调节作用并初步探讨以上调节作用和影响的分子机制。
实验方法:
1.运用双荧光素酶报告基因系统以及western blot等方法检测生长因子刺激或者瞬时转染Ras对p28~(GANK)基因表达水平的影响;
2.应用报告基因系统结合ERK与PI3K信号通路抑制剂确定生长因子与Ras对p28~(GANK)表达的调节通路;
3.利用报告基因系统、real-time PCR以及western blot等方法观察瞬时转染Akt表达质粒对p28~(GANK)基因表达的影响;
4.多种肝癌细胞系中高表达p28~(GANK),观察p28~(GANK)对p-Akt的影响;
5.通过Luciferase报告基因质粒pGL-OT(含有β-catenin/TCF4复合物特异结合区)及对照报告基因质粒pGL-OF与不同剂量p28~(GANK)质粒细胞共转染实验,确定p28~(GANK)对β-catenin/TCF4转录活性的影响;
6.运用western blot及核浆分离技术,检测HEK293细胞瞬转p28~(GANK)后,β-catenin在胞核胞浆中的分布变化;应用腺病毒介导的RNA干扰技术下调肝癌细胞系中p28~(GANK)的表达水平,探讨其对β-catenin转录活性的影响;
7.应用western blot和免疫组织化学方法检测临床肝癌组织标本中β-catenin、c-Myc、cyclinD1表达水平与p28~(GANK)蛋白表达水平之间的关系;
8.利用双荧光素酶报告基因系统研究差异表达p28~(GANK)对其自身报告基因转录活性的影响;运用western blot检测外源性p28~(GANK)表达增加对内源性p28~(GANK)表达的影响。
结果:
1.肝细胞生长因子、表皮生长因子刺激和Ras表达质粒瞬时转染都能够明显上调饥饿处理后的HEK293和HepG2细胞中p28~(GANK)的表达水平;
2. PI3K/AKT通路介导上述刺激对p28~(GANK)的上调作用,而且瞬时转染Akt表达质粒能够上调p28~(GANK)转录及蛋白水平的表达;而且HCC组织样品中p-Akt水平与p28~(GANK)的表达存在正相关性;
3. Huh7、L02及HepG2细胞中瞬时转染p28~(GANK)质粒均可显著增强AKT蛋白磷酸化水平;
4. HEK293细胞中瞬时转染p28~(GANK)表达质粒可提高β-catenin入核蛋白水平,并在一定程度上稳定细胞中β-catenin总蛋白水平,增强β-catenin/TCF4转录活性;
5.报告基因、RT-PCR或western blot实验证明瞬时转染表达β-catenin和c-Myc都能够在转录和蛋白水平上调p28~(GANK)表达;
6. Western blot和免疫组化实验分析表明p28~(GANK)的蛋白水平在肝癌组织样品中与β-catenin、c-Myc和cyclinD1呈同向性表达;
7.在HEK293和Hep3B中p28~(GANK)正向调节自身表达。
结论:
本研究发现生长因子刺激或Ras激活可以上调肝癌癌蛋白p28~(GANK)的表达,其中PI3K/AKT信号通路具有重要作用。Akt、β-catenin和c-Myc的上调表达都能够促进p28~(GANK)的表达。进一步实验表明上调的p28~(GANK)可以反过来进一步促进β-catenin蛋白的稳定及其核内定位从而上调β-catenin/TCF4转录活性,而且p28~(GANK)过表达能够增强AKT蛋白磷酸化水平。上述结果支持我们提出的p28~(GANK)以正反馈方式调节β-catenin信号通路的科学推测,为深入理解p28~(GANK)影响肝癌发生发展的信号调控机制提供了依据。
Part I p28~(GANK) Enhances Oxidative Stress Tolerance of HCC cell lines by Promoting Detoxifying Enzymes Expression
Tolerance to oxidative stress of cancer cells determines their survival advantage with active proliferation during the pathogenesis and development of hepatocelluar carcinoma (HCC), and also cancer cells' drug resistance in chemotherapy. So finding out critical molecular targets that regulate the oxidative stress tolerance of HCC cells is significant for HCC therapy. p28~(GANK) is one onco-protein highly expressed in HCC. Our previous study revealed that interfering p28~(GANK) expression in HCC cell lines induces apoptosis via elevating intracellular ROS level. In the present study, we investigated the expression of p28~(GANK) under oxidative stress conditions and examined its protential role in modulating HCC cells' tolerance to oxidative stress. Our results confirmed that p28~(GANK) expression is upregulated both in H_2O_2-induced moderate oxidative stress in HCC cell lines and in DEN-induced oxidative stress in rat HCC model. Furthermore, we demonstrated that p28~(GANK) protects HCC cells from oxidative stress-induced cell death probably through enhancing ROS-detoxifying enzymes expression, resulting in reduction of intracellular ROS accumulation. In addition, p28~(GANK) may pomotes cell survival via activating PI3K/AKT pathway under oxidative stress.
Part II The oncoprotein p28~(GANK) establishes a positive feedback loop inβ-catenin signaling
GANKYRIN (also known as PSMD10 or p28~(GANK)) is a novel oncoprotein highly expressed in hepatocellular carcinoma (HCC) and interacts with multiple proteins leading to degradation of tumor suppressor proteins, Rb and p53. p53 was reported to downregulateβ-catenin, but whether p28~(GANK) is involved in regulation ofβ-catenin is elusive. Herein we report that both growth factors and Ras upregulated p28~(GANK) expression thrsough the activation of the PI3K-AKT pathway. The induced p28~(GANK) expression subsequently enhanced the transcription activity ofβ-catenin, and this effect was observed in p53-deficient cells, suggesting a p53-independent mechanism for p28~(GANK) regulation ofβ-catenin activation. p28~(GANK) overexpression also increased release of freeβ-catenin into cytoplasm. Interestingly, exogenous expression of p28~(GANK) resulted in elevated expression of the endogenous protein. The present study also suggested that bothβ-catenin and c-Myc were transcriptional activators of p28~(GANK) and p28~(GANK) overexpression correlated with c-Myc, cyclin D1 andβ-catenin was detected in primary human HCC. In aggregate, these results suggest a positive feedback loop for p28~(GANK) expression, which may play a critical role in tumorigenesis and progression of HCC.
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