【作者】 文文；

【导师】 王红阳；

【作者基本信息】 第二军医大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 第一部分HIF-1负调控cyclin D1的分子机制及其在肺癌化疗抵抗中的作用研究研究背景和目的哺乳动物细胞可以通过改变代谢状态和生长速度来适应环境中氧浓度的变化,其中最常见反应就是生长抑制。低氧可以引起多种细胞的周期阻滞,但具体机制仍未阐明,近年来研究发现低氧诱导因子(Hypoxia-Inducible Factor-1,HIF-1)是介导这一过程的重要分子。HIF-1以异源二聚体的形式存在,由α亚单位(HIF-1α)和β亚单位(HIF-1β)组成。常氧条件下,HIF-1α极易被降解,其降解途径是泛素—蛋白水解酶复合体;低氧条件下,HIF-1α降解过程被阻断,导致HIF-1α积累,并与HIF-1β结合形成活性形式,影响多种靶基因表达,参与调节血管形成、铁代谢、葡萄糖代谢、细胞增殖和存活等生理过程。HIF-1靶基因结构上都包含有低氧反应元件(Hypoxia Response Element,HRE),HRE是指被调控基因的启动子或增强子中包含一段特定的核苷酸序列,其核心序列为5’-RCGTG-3’。活化的HIF-1与其结合位点结合后,在靶基因的转录起始部位形成一个转录起始复合物,从而调节靶基因的转录。细胞周期是一种非常复杂的过程并受到精细的调节,有大量调节蛋白参与其中。此过程的核心是细胞周期依赖性蛋白激酶(Cyclin Dependent Kinases, CDKs),CDK的激活又依赖于另一类呈细胞周期特异性或时相性表达的细胞周期蛋白(cyclins),其中cyclin D1就是一种非常重要的周期蛋白。Cyclin D1基因属于细胞周期素家族中的一员,基因定位于染色体l1q13,编码由295个氨基酸组成的蛋白质。Cyclin D1可与细胞周期索依赖激酶CDK4组成Cyclin D1/CDK4复合物,使Rb磷酸化,从而释放出转录因子E2F,促进细胞周期由G1期到S期的转变。机体中的细胞时常受到低氧的刺激,而在肿瘤细胞特别是实体瘤中,缺氧更是普遍存在的现象。低氧条件下,细胞中产生的HIF-1调节靶基因转录,引起一系列细胞对缺氧的反应,具有重要的病理生理学意义。肿瘤组织的缺氧被认为与肿瘤对放疗、化疗耐受及恶性进展、远处转移密切相关。近年来发现HIF-1是肿瘤治疗中重要的靶点分子,可能参与了实体肿瘤对化疗药物的耐药机制。我们预实验的结果发现HIF-1对cyclin D1存在负调控作用,而且通过序列分析,在cyclin D1的启动区中发现了HIF-1结合位点,本研究旨在阐明HIF-1对cyclin D1的调控机制,观察这种调控作用对肿瘤细胞生长和化疗敏感性的影响,加深我们对肿瘤生物学的认识,为研发新的肿瘤治疗药物和方法提供理论依据。实验方法:一、利用外源刺激物或低氧处理,观察HIF-1对cyclin D1的负调控作用1.外源刺激物诱导或低氧处理使HIF-1活化后,利用报告基因、RT-PCR和western-blot检测正常细胞和肿瘤细胞中cyclin D1表达的变化;2.应用HIF-1基因敲除细胞验证在正常细胞中HIF-1对cyclin D1的调控作用;3.探明在生理条件下,肿瘤细胞中过度表达HIF-1对cyclin D1的影响;4.利用HIF-1的显性负性突变体(DN-HIF)抑制HIF-1活化,对上述的结果进行验证。二、HIF-1对肿瘤生长的影响1.建立稳定表达DN-HIF的A549和Beas-2B细胞系;2.利用流式细胞分析和CCK-8生长曲线测定方法,观察抑制HIF-1活性对肿瘤细胞的细胞周期和增殖的影响;3.将上述肿瘤细胞接种实验动物后,观察肿瘤的生长情况;4.利用免疫组化方法验证荷瘤鼠肿瘤中HIF-1与cyclin D1表达的关系。三、HIF-1对cyclin D1调控作用的分子机制1.构建启动区中HRE突变的cyclin D1报告基因质粒,分析HIF-1对cyclin D1的调控是否为直接作用;2.进行ChIP实验,进一步明确HIF-1与cyclin D1 promoter间的直接相互作用;3.构建HDAC的RNA干扰质粒,并利用HDAC抑制剂TSA,分析HDAC在HIF-1负调控cyclin D1机制中的作用。四、HIF-1在肺癌细胞化疗敏感性中的作用1.利用TUNNEL方法和流式细胞仪检测,观察HIF-1在周期特异性化疗药物5-FU诱导的肺癌细胞凋亡中的作用;2.研究HIF-1对cyclin D1的调控作用对肿瘤细胞化疗敏感性的影响。结果:1.在支气管上皮细胞Bea-2B中,砷(Arsenite)能够导致HIF-1α的蓄积,并诱导下游靶基因VEGF表达,上调HRE报告基因水平,同时也能够诱导cyclin D1表达;2.稳定表达HIF-1显性负性突变体的支气管上皮细胞和肺癌细胞系中,砷诱导的cyclin D1的转录和表达较对照组细胞明显增强,共转DN-HIF和cyclin D1 eGFP报告基因的293T细胞中eGFP荧光强度高于对照细胞;3.流式细胞检测发现,稳定表达HIF-1显性负性突变体的肿瘤细胞的G1/S期转化较对照细胞明显增快,促进肿瘤细胞增殖。荷瘤小鼠体内实验结果也发现HIF-1与cyclin D1表达呈负相关;4.报告基因结果显示,低氧条件下,HIF-1α积累,并能够显著抑制cyclin D1报告基因活性,同时HIF-1α与cyclin D1蛋白表达呈负相关。5.染色质免疫共沉淀实验证实,HIF-1α通过与cyclin D1启动区的低氧反应元件直接结合发挥抑制作用,而砷刺激对ΔHRE cyclin D1报告基因的诱导显著高于对照组;6.TSA能够阻断外源表达HIF-1对cyclin D1的抑制,而且干扰HDAC 7也可以明显削弱这种抑制作用;7.A549细胞中外源表达DN-HIF能够显著增加5-FU引起的细胞凋亡,而共转sicylin D1表达质粒后凋亡细胞比例明显降低。同时,外源表达DN-HIF对细胞周期非特异性药物Etoposide引起的细胞凋亡没有明显的影响。结论:1.在正常肺上皮细胞和肿瘤细胞中,常氧条件和低氧条件下,低氧诱导因子-1(hypoxia inducible factor-1, HIF-1)均能够负调控周期素D1(cyclin D1);2. HIF-1可以直接与cyclin D1启动区中的HRE相互作用,起到负调控的作用,该调控作用需要组蛋白去乙酰化酶-7(HDAC7)的参与,TSA处理和干扰HDAC7均能抑制这种负调控;3. HIF-1负调控cyclin D1能够引起肺癌细胞A549周期阻滞,抑制细胞的增殖,在荷瘤小鼠体内也能观察到HIF-1与cyclin D1的负相关,这可能是肿瘤细胞逃避放、化疗的机制之一;4. A549细胞中外源表达DN-HIF能够显著增加细胞周期特异性化疗药物5-FU引起的细胞凋亡,而干扰cyclin D1后5-FU引起的细胞凋亡被明显抑制。这为我们理解肿瘤化疗抵抗的机制和开发新的治疗药物提供了新的思路。第二部分Cyclin G1诱导上皮间质转化促进肝癌转移研究背景和目的肝癌是恶性程度极高、预后极差的肿瘤。全世界半数左右的肝癌病人集中在中国,其致死率居我国所有肿瘤的第二位。临床肝癌诊疗中存在的早期诊断难、复发转移率高、治疗缺乏针对性、源头创新药物和治疗手段少等难点问题的解决仍没有取得很大进展。统计资料表明,肝癌术后5年复发率接近60%,肝癌治疗后的复发转移是目前影响肝癌疗效的重要因素。国内外对复发再切除持肯定态度并以此作为复发治疗的首选,但对个体而言,复发再切除往往因病人的全身情况差等原因而受到限制。临床上应用的抗复发措施颇多,如TACE、全身化疗、放疗和中医中药等,但效果均不理想。复发转移的机理不明是临床缺少特效新药和有效治疗手段的主要原因。肝癌复发转移是一个十分复杂的问题,国内外研究发现不少的癌基因、生长因子等与肝癌侵袭性相关,多种细胞外基质成分也有重要作用,而且可能与原发瘤的基因特性有关。但是肝癌复发转移的机制仍然没有完全阐明,下一步的重点将是如何早期预测肝癌的复发转移,并尽早施行针对性的抗转移复发治疗。由于传统的临床预后指标有其自身的局限性,尚不能完全反映肝癌的生物学特性。为了进一步指导肝癌的诊断、治疗和预后的判断,寻找其它有效的分子生物学指标一直以来都是肝癌研究的热点。细胞周期蛋白G是cyclins家族成员之一,目前发现cyclin G主要有2种亚型cyclin G1和cyclin G2。Cyclin G1与其他的周期蛋白不同,其N端有一典型的细胞周期蛋白盒,但没有降解盒或PEST序列,也没有找到相应的细胞周期蛋白依赖性激酶,其生物学功能尚未完全阐明,有证据表明cyclin G1可能参与DNA损伤修复、细胞周期调控、凋亡等细胞生物学过程,近来研究发现cyclin G1与肿瘤的关系密切,在多种肿瘤中都存在着异常表达,可能对肿瘤的发生发展起到了促进作用。已证实cyclin G1是p53的靶基因,同时又能通过Mdm2负反馈调节p53,cyclin G1敲除细胞中p53水平升高,由于p53是一种公认的抑癌基因,cyclin G1与p53的动态平衡可能对维持细胞的正常表型起到了重要的作用。最新的动物实验研究也发现cyclin G1缺失能够降低化学致癌物诱导的肝癌发生率,提示cyclin G1与肝癌的关系密切。但目前尚未见任何关于cyclin G1与肝癌预后的研究报道。因此,本课题分析肝癌中cyclin G1的表达及其临床意义,同时初步探讨了cyclin G1在肝癌转移中的作用及可能的机制,旨在为肝癌复发转移的防治提供新的分子标志物和干预靶点。实验方法:1.利用RT-PCR、western-blot和免疫组织化学方法检测肝癌细胞系或者肝癌临床组织样品中cyclin G1的表达情况;2.利用慢病毒系统建立cyclin G1稳定高表达的细胞株L02/cyclin G1、SMMC-7721/cyclin G1和HepG2/cyclin G1及其相应的对照细胞株;3.侵袭浸润实验:比较SMMC-7721/cyclin G1和HepG2/cyclin G1与其对照组SMMC-7721/GFP和HepG2/GFP在侵袭浸润特性上的差异;4.划痕损伤实验:比较SMMC-7721/cyclin G1和HepG2/cyclin G1与其对照组SMMC-7721/GFP和HepG2/GFP在迁移动力学功能上的差异;5.生长曲线:比较L02/cyclin G1、SMMC-7721/cyclin G1和HepG2/cyclin G1及其对照细胞系在分裂增殖上的差异;6.动物试验:比较SMMC-7721/cyclin G1细胞系与其对照细胞在裸鼠体内成瘤和转移特性的差异;7.形态学观察,免疫组织化学等方法评价cyclin G1对肝癌细胞株上皮间质转化(EMT)的影响,并检测EMT相关特征分子和干细胞标志物的表达变化。利用流式细胞和经典信号通路的研究方法,初步探讨cyclin G1影响肝癌转移特性的分子机制。结果:1.cyclin G1在肝癌细胞系和肝癌组织样品中均呈高表达,致癌物刺激能够上调L02细胞系中cyclin G1的mRNA水平;2.利用慢病毒系统,成功建立了cyclin G1外源性高表达稳定细胞株L02/cyclin G1、SMMC-7721/cyclin G1和HepG2/cyclin G1及其相应的对照细胞株;3.cyclin G1外源性高表达对肝癌细胞系SMMC-7721和HepG2生物学功能的影响,侵袭浸润实验和划痕损伤实验显示,与对照组相比SMMC-7721/cyclin G1和HepG2/cyclin G1的侵袭运动能力明显提高;4.生长曲线结果显示L02/cyclin G1、SMMC-7721/cyclin G1和HepG2/cyclin G1及其对照细胞系在分裂增殖上没有明显差异;5.SMMC-7721/cyclin G1细胞在体外培养环境中出现间质细胞形态特征,动物体内实验结果显示,外源性cyclin G1高表达,能够显著提高SMMC-7721在裸鼠肝内播散成瘤的能力,免疫组织化学结果显示间质细胞特征性分子vimentin与cyclin G1呈正相关;6.用Western-blot方法检测肿瘤转移相关的一些信号通路,结果发现cyclin G1可以活化PI3K/AKT和MAPK信号通路;Realtime-PCR实验表明,cyclin G1能够上调Snail和Twist,并引起下游间质细胞标志物FN1转录增加,而上皮细胞特征分子E-cad、Desmoplakin和Zo-1下调;7.realtime-PCR结果显示SMMC-7721 cyclin G1细胞株中干细胞相关分子Oct4、ABCG2、Bmi 1和Nanog的表达显著高于对照组。流式细胞检测发现外源性cyclin G1高表达能够显著提高HepG2细胞系中CD133分子阳性细胞的百分比。结论:1.Cyclin G1在肝癌细胞系和临床组织样品中普遍呈现中高表达,提示其与肝癌发生发展有密切的相关性;2.Cyclin G1在肝癌中不仅与肝癌发生相关,还可能作为一种肿瘤进展基因起作用,可以影响肝癌细胞的侵袭转移特性,从而提高肝癌的恶性程度;3.Cyclin G1可能通过作用于PI3K/AKT信号通路对EMT的相关分子进行调控,促进上皮间质转化,甚至获得干细胞潜能,最终影响肝癌细胞的浸润转移能力;4.Cyclin G1有可能成为判断肝癌患者进展及预后的一个新分子标志物,为肝癌的预后判断及对其转移的有效预防和治疗提供新的分子靶点。更多还原

【Abstract】 Part I: Suppression of Cyclin D1 by Hypoxia-Inducible Factor-1 via Direct Mechanism Mediates Chemoresistance in Lung Cancer CellsHypoxia-inducible factor (HIF) and cyclin D1 are key mediators of cell growth and proliferation in both normal and cancer cells. However, the interrelation between HIF and cyclin D1 remains unclear. Although genetic studies from the mouse HIF-1αnull cells had strongly indicated that HIF-1αis required for hypoxia-induced cell cycle arrest, the mechanism underlying hypoxia-induced cell cycle arrest remains undetermined so far. Sequence analysis of the cyclin D1 promoter revealed the putative binding sites for the HIF-1. Therefore, it is important to investigate whether HIF-1 regulates cyclin D1 expression or not, and if it does, what will be the detailed mechanism and subsequent outcome.In current study, arsenite which is regarded as a heavy metal and can mimic hypoxia condition by enhancing the stability of HIF-1αprotein was used to study the correlation between HIF and cyclin D1. To test whether HIF-1 was able to regulate cyclin D1 expression, dominant negative mutant of HIF-1α(DN-HIF) stable transfectants were established. Cyclin D1 expression in DN-HIF stable transfectant was significantly increased, indicating the suppression of cyclin D1 by HIF-1 in Beas-2B cells exposed to arsenite. To further confirm this finding, HIF-1αknockout MEF cells(HIF-1α-/-) was used in current study and the results showed that cyclin D1 induction by arsenite in HIF-1αknockout cells was notably enhanced compared with that in WT cells. In addition, we found that impairment of HIF1-αincreased cyclin D1 expression in A549 pulmonary cancer cells, and which in turn promoted G1/S cell cycle transition and cell proliferation. Furthermore, cyclin D1 expression was increased in subcutaneous xenograft of DN-HIF stably-transfected A549 cells in nude mice compared with that of control cells. ChIP assay revealed that HIF was able to directly bind to the promoter region of cyclin D1, which indicates the negative regulation of cyclin D1 by HIF was through a direct mechanism. A significant increase of cylin D1 promoter activation by arsenite was observed inΔHRE cyclin D1 reporter transiently transfected A549 cells compared with wide-type cyclin D1 reporter transfected cells, which indicates HIF negatively regulates cyclin D1 transcription via the interaction with cyclin D1 promoter. It has been reported that HDAC7 was able to interact with HIF-1αand mediated the expression of HIF downstream target gene. Not only TSA pretreatment but also introduction of shRNA of HDAC7 significantly antagonized the suppression of cyclin D1 by HIF revealing that HDAC7 was required in HIF-mediated cyclin D1 down-regulation. Moreover, we found the increased HIF-1αexpression or decreased cyclin D1 expression both led to the chemoresistance of A549 cells upon 5-FU treatment, suggesting HIF-1 over-expression associated chemoresistance might be due to, at least partially, the negative regulation of cyclin D1.Base on the data achieved in present study, we conclude that the negative regulation of HIF-1αon cyclin D1 is through its interaction with HRE sequence in cyclin D1 proximal promoter, which also requires HDAC7 involvement. HIF-1 induced G0/G1 phase arrest may account for the resistance of cancer cells to some certain chemotherapeutic agents that target DNA synthesis in S phase.Elevated expression of HIF-1 is usually detected in various solid tumors including colon, breast, prostate, and lung cancers, and is associated with resistance to therapies and poor prognosis in head and neck cancer, ovarian cancer and oesophageal cancer. The maximal therapeutic effects are likely to be achieved by treating patients with several different types of anticancer drugs, just as we use“cock-tail therapy”for infections and hypertension. Given that HIF-1 is likely to be one of the most valuable therapeutic targets, the possible clinical implications of targeting HIF-1 in cancer still need to be fully defined. Part II: Cyclin G1 Facilitates Liver Cancer Metastasis via Promotion of Epithelia Mesenchymal TransitionLiver cancer is the sixth most common cancer worldwide in terms of number of cases (626,000 or 5.7% of new cancer cases) but because of the rather poor prognosis, the number of deaths is almost the same (598,000). It is therefore the third most common cause of death from cancer. Survival rates are 3% to 5% in cancer registries for the United States and developing countries. Distant metastasis is one of the main reasons for the death of liver cancer. It is very important to find out the patients who belong to the high-risk group of recurrence and metastasis, and give them sufficient adjuvant therapy to improve the survival. Traditional clinical prognostic indexes fail to reflect the biological feature of liver cancer completely. With the development of molecular biology, it has become a hotspot in the field of liver cancer research that seeking new indexes to direct the diagnosis or treatment and predict the prognosis.Cyclin G1 belongs to a subgroup of cyclins, which also includes cyclin G2 and cyclin I. Functional cyclin-dependent kinase partners for these cyclins have not been described, and the biologic function(s) of this subgroup remain to be firmly established. In fact, cyclin G1 deregulation is associated with genomic instability and increased cyclin G1 levels have been described in colorectal cancer, breast cancer, and leiomyoma. Moreover, experimental evidences achieved inform cancer cell lines and tumor xenografts have shown that suppression of cyclin G1 results in the inhibition of tumor growth through the reduction of proliferation and induction of apoptosis. In experimental hepatocarcinogenesis, loss of cyclin G1 is associated with a significantly lower tumor incidence after carcinogenic challenge and cyclin G1–null hepatocytes enter S phase at a lower rate. Cyclin G1 is transcriptionally activated by p53 and p73, and, in turn, it negatively regulates p53 family proteins via a feedback regulation. Taken together, these data suggests a link between cyclin G1 and liver carcinogenesis and progression.At present, the relationship between cyclin G1 and prognosis of liver cancer has been poorly reported and no functional study on cyclin G1 in late stage of liver cancer was reported. Therefore, in this study we investigated the expression of cyclin G1 in liver cancer, as well as the possible functions of cyclin G1 in liver cancer metastasis and the underlying molecular mechanism.In the results of this study, we showed that cyclin G1 mRNA level in liver cancer cell lines is higher than that in normal liver cells, and could be induced by carcinogens such as DEN. By immunohistochemical analysis, the high expression of cyclin G1 protein was detected in all of the paraffin embedded specimens of liver cancer and mainly located in tumor cell nuclei. Western blot assay revealed 62.5% cases with high expression of cyclin G1 in protein extracts of liver cancer tissue versus the peri-tumor tissue. To investigate the effect of cyclin G1 on behavior of tumor cells, lenti-virus was utilized to establish cyclin G1 overexpression cell lines. There was no significant difference in proliferation index of cyclin G1 stable transfectants and the control cells. However, results from cell invasion assay and wound healing assay demonstrated that over-expression of cyclin G1 enhances the migration and invasion abilities of SMMC-7721 and HepG2 cells. Importantly, more intrahepatic metastatic nodes were observed in the nude mice inoculated with SMMC-7721 cyclin G1 mass cells after 6 weeks than those inoculated with control cells via spleen. To clarify the underlying molecular mechanism we tested several signaling pathways which are closely related to tumor metastasis. We found that cyclin G1 could promote EMT through PI-3K/AKT cascade and further triggered the dedifferentiation of liver cancer cells which in turn facilitates the invasion and metastases of tumor cells.Taken together, in present study, we observed cyclin G1 is over-expressed in liver cancer tissues and Demonstrated that cyclin G1 regulates EMT by activation of AKT pathway, which is responsible, at least partially, for the invasion and metastasis of liver cancer. We suggest that cyclin G1 may be a valuable marker for assessing the prognosis of liver cancer, and can be used as therapeutic target for preventing liver cancer from metastasis.更多还原