【作者】 曾季平；

【导师】 崔行； 贾继辉； Dawei Xu；

【作者基本信息】 山东大学 ， 生化与分子生物学， 2009， 博士

【摘要】 胃癌是最常见的恶性肿瘤之一,在全世界所有与癌症相关的死亡当中,胃癌居于第二位。研究者们已证明幽门螺杆菌感染与胃癌发生之间的密切关系,现一般认为,幽门螺杆菌感染首先引起慢性胃炎的发生,而慢性胃炎是胃组织向癌症转化的病理生理异常的前兆,幽门螺杆菌正是以这种方式参与了胃癌的早期发生过程。但是,这些癌前病变仅仅在部分感染幽门螺杆菌的人群中出现,且并非必然发展至侵袭性肿瘤阶段。虽然其具体的分子机制还不是十分清楚,但是已经知道个体的遗传特征、细菌毒力、环境以及其他多种因素均不同程度地影响着幽门螺杆菌感染的致癌性可能。另外,由于缺乏可靠的早期诊断指标以及有效的干涉手段,目前胃癌的预后仍然不容乐观。因此,仍需要大量的科学研究来进一步探索胃癌的发病机理,寻找有效的生物学标志物以及新的治疗靶点。调节细胞增殖与生存的关键分子发生异常表达而导致的细胞无限增殖是包括胃癌在内的所有恶性肿瘤最重要的特征。新近的一些研究已经证实,细胞衰老可保持不可逆的细胞生长停滞状态,是一种极具潜力的肿瘤抑制机制。也有证据表明诱导癌细胞衰老将有利于增强化疗药物及其他抗肿瘤治疗方法的疗效。目前研究证明,端粒酶逆转录酶（hTERT）是端粒酶发挥作用的限速成分,通过端粒酶逆转录酶的诱导而使端粒酶激活,这是肿瘤细胞越过衰老的屏障发生恶性转化的关键手段。此外,作为对细胞寿命起关键调控作用的p53和p16,是两个重要的肿瘤抑制因子,他们的功能性失活将阻断细胞的内源性衰老进程,进而从另一方面激活了肿瘤细胞无限增殖的潜能。虽然目前已经取得了这些进展,我们却仍未完全掌握肿瘤细胞保持无限增殖能力的确切机制以及是否尚存在其他的致癌因素参与胃癌及其他肿瘤细胞的衰老逃逸。因此在本研究中,我们选取细胞周期转录促进因子FoxM1和新型组蛋白去甲基化酶RBP2作为靶点,通过研究这两种蛋白在胃癌发生中的作用及与细胞衰老的关系,为寻找新的监测和防治胃癌的作用位点提供理论基础。Forkhead Box M1（FoxM1）转录因子是有丝分裂中的关键正调控蛋白,已被证明参与多种癌症的发生与发展过程。本研究首次揭示了FoxM1在胃癌细胞中的作用及在人体原发性胃癌标本中表达情况。实验证明,在多种胃癌细胞株中均存在FoxM1的表达。当利用RNAi技术抑制胃癌细胞株中FoxM1表达时,有明显的细胞衰老现象,肿瘤细胞的生长及克隆形成能力受到抑制。这一过程与P53及P16信号途径无关。当抑制FoxM1在细胞中的表达时,其目的调控基因c-myc和skp2的表达也随之下调,同时CDK的抑制因子p27^kip1表达上升。当利用特异性的siRNA抑制p27^kip1活性时,即使抑制FoxM1的表达,细胞衰老现象也不明显,肿瘤细胞的生长和克隆形成能力有所恢复。在这一衰老过程中,在细胞癌变过程中起关键作用的核蛋白酶端粒酶的重要组分端粒酶逆转录酶（hTERT）的mRNA表达水平及蛋白活性也明显下降。为进一步证明FoxM1在胃癌发生发展中的作用,本实验还随机选取了42对人原发性胃癌及对应的癌旁正常组织进行检测。RT-PCR,实时定量PCR及免疫组化检测结果均表明:在胃癌标本中有39/42个（93%）标本检测到FoxM1表达,但在正常标本中只检测到8个（19%）;最终共有38/42对（90%）标本中FoxM1在胃癌组织中高表达。综上所述,FoxM1在胃癌中被显著活化,它的活性对维持胃癌细胞的生长是必不可少的。在胃癌细胞中如果抑制FoxM1的表达,可导致不依赖于P53和P16信号途径的细胞衰老过程。这一过程受p27^kip1及hTERT等其他相关分子的调控。通过以上实验,可得到如下结论:（1）FoxM1在胃癌组织中普遍高表达,但在癌旁正常组织中几乎不表达;（2）在胃癌细胞中利用特异性siRNA抑制FoxM1的表达后,可明显抑制肿瘤细胞的生长和克隆形成能力,机制在于可诱发细胞衰老;（3）在胃癌细胞中抑制FoxM1表达所致的细胞衰老过程不依赖于p53和P16信号通路,而是部分依赖于P27^KIP1的激活。但在胃癌细胞中抑制P27^KIP1的活性只能部分恢复抑制FoxM1表达所导致的胃癌细胞克隆形成减少及细胞衰老,因此存在其他的信号转导调控机制;（4）在FoxM1受到抑制的胃癌细胞中,FoxM1关键下游因子c-Myc的表达也受到显著抑制;同时,c-Myc的下游靶基因hTERT的表达及端粒酶的活性也随之下降。组蛋白修饰失衡和变异的染色体重塑在癌症发生、发展过程中起着重要作用。其中一种重要的方式是组蛋白修饰酶过度表达。RBP2作为一种新发现的作用于组蛋白3上第四个赖氨酸（H₃K₄）三或二甲基化的组蛋白去甲基化酶,参与癌症发生发展,其在胃癌中是否起到重要作用值得探讨。在随机选取的42对人原发性胃癌及对应的癌旁正常组织标本中,通过RT-PCR,实时定量PCR及免疫组化实验证明在胃癌组织中RBP2的表达量明显高于正常对照组织（30/42,71.5%）。同时,RBP2在多种胃癌和宫颈癌细胞株中均有表达。利用RNAi技术抑制RBP2在胃癌细胞和宫颈癌细胞中的表达可诱导明显的细胞衰老现象;肿瘤细胞的生长与克隆形成同时受到抑制。这一过程有p21^CIP2,p27^kip1和p16^ink4a等CDK的抑制剂（CDKI）参与其中。同时,双荧光素酶活性检测证明,在胃癌细胞中利用特异的siRNA抑制RBP2表达或使用表达质粒高表达RBP2可使p21^CIP2,p27^kip1和p16^ink4a的启动子活性上升或下降。ChIP法检测表明RBP2在胃癌细胞中结合于p21^CIP2,p27^kip1和p16^ink4a启动子区,当RBP2被抑制后,结合解除,则H3K4的甲基化程度显著上升。p21^CIP2和p27^kip1特异性的siRNA可使抑制RBP2表达导致的胃癌和宫颈癌细胞衰老被部分解除,肿瘤细胞的增殖和克隆形成有所恢复。综上所述,可得以下结论:（1）RBP2在胃癌组织中普遍高表达;（2）在胃癌细胞及宫颈癌中利用特异性siRNA抑制RBP2的表达后,可明显抑制肿瘤细胞的生长和克隆形成能力,机制在于可诱发细胞衰老;（3）这一过程与CDKIp16^ink4a、p21^CIP2和p27^kip1的激活相关。RBP2可直接与p16^ink4a、p21^CIP2和p27^kip1的启动子结合来影响基因的表达;（4）在癌细胞中抑制p21^CIP2或P27^KIP1的活性能部分恢复抑制RBP2表达所导致的胃癌细胞克隆形成减少及细胞衰老。本研究深入探讨了FoxM1和RBP2在维持胃癌细胞增殖,抑制细胞衰老及生长停滞的分子机制,揭示了其在胃癌发生、发展的关键作用。研究结果表明抑制FoxM1和RBP2在胃癌中的表达是抗肿瘤的可能机制之一。FoxM1和RBP2可作为胃癌监测与治疗的潜在作用靶点。在胃癌的诊断与治疗中有广泛的应用前景。更多还原

【Abstract】 Gastric cancer is one of the most common malignancies and second leading cause of cancer-related death worldwide. The intimate relationship between Helicobacter pylori infection and gastric cancer has long been established and it is proposed that Helicobacter pylori infection is involved in an early stage of gastric cancer pathogenesis by causing a chronic gastritis, the precursor to all the patho-physiologic abnormalities characteristic of gastric carcinogenesis. However, these pre-malignant lesions only develop in a proportion of infected subjects and do not necessarily progress into invasive cancers. The host genetic, bacterial virulence, environmental and many other factors are suggested to significantly affect the oncogenic process initiated by Helicobacter pylori infection, but the underlying molecular mechanism is largely unclear. Additionally, because of lacks of reliable early diagnostic markers and efficient intervention approaches, the prognosis of gastric cancer remains poor. Therefore, better defining the pathogenesis of gastric cancer, looking for useful biomarkers, and exploring novel therapeutic targets for treatment are urgently demanding tasks.A hallmark of cancers including gastric cancer is unlimited cellular proliferation due to the aberrant expression of key factors regulating cell proliferation and survival. Recent studies have shed light on cellular senescence, an irreversible cell growth arrest status, as a potent tumor suppression mechanism. Evidence has also accumulated that induction of senescence contributes to the treatment efficacy of chemotherapeutic agents and other anti-cancer strategies. It is known that activation of telomerase through induction of its rate-limiting component telomerase reverse transcriptase （hTERT） is a critical strategy that erases senescence barrier during malignant transformation. Moreover, functional inactivation of the tumor suppressors p53 and p16, the key regulators of cellular life-span, disrupts intrinsic senescence program, thereby contributing to the infinite proliferation potential of cancer cells at another layer. Despite such general knowledge, however, it remains incompletely understood how exactly sustained cancer cell proliferation is maintained and whether other oncogenic factors are involved in the prevention of senescence of gastric and other cancer cells. In this research, FoxM1 and RBP2 were selected to study the role of them in gastric cancer and the relation with cell senescence.The Forkhead Box M1 （FoxM1） transcription factor, a master regulator of mitotic gene expression, promotes the development and progression of several types of malignancies. In the present study, we determined the functional role of FoxM1 in gastric cancer cells and its expression in primary gastric cancers. FoxM1 was expressed in all examined gastric cancer cell lines and when it was knocked-down, impaired clonogenicity and cellular senescence occurred independently of p53 and p16 status. Following FoxM1 depletion, its target genes c-MYC and SKP2 were substantially down-regulated, which was coupled with the accumulation of the CDK inhibitor p27kip1. Importantly, the FoxM1 inhibition-mediated cellular senescence and clonogenic defect was substantially prevented by the abolishment of p27kip1 induction. Telomerase reverse transcriptase （hTERT）, the key component of telomerase, a nucleoprotein enzyme essential for cellular immortalization, was also inhibited in the FoxM1-depleted gastric cancer cells. Moreover, the over-expression of FoxM1 was observed in 39 of 42 tumor specimens derived from patients with gastric cancer. Collectively, the FoxM1 gene is aberrantly activated in gastric cancer and is required for sustained proliferation of the cancer cells. The FoxM1 deficiency triggers p53 and p16-independent senescence of cancer cells by regulating the expression of p27kip1 and other targets. The findings provide mechanistic insights into roles of FoxM1 in pathogenesis of gastric cancer and FoxM1 may thus be implicated in the diagnosis and treatment of gastric cancer.The aberrant expression of histone-modifying enzyme has long been implicated in oncogenesis. However, it is unclear whether RBP2, a newly identified histone demethylase, involved in the pathogenesis of carcinoma including gastric cancer. We determined RBP2 expression in gastric cancer and its functional role in cancer cells. Cancerous and matched normal specimens from 42 patients with gastric cancer were analyzed for RBP2 expression using quantitative real-time PCR （QRT-PCR） and immunohistochemistry. RBP2 and other gene expression in cell lines was assessed using QRT-PCR and immunoblotting, and knocked down with specific small interference RNA. Clonogenesis and cellular senescence were determined using foci formation and Senescence associatedβ-Galactosidase staining. The promoter activity was determined by luciferase reporter assay. Chromatin immunoprecipitation was used to examine RBP2 and methylated histone H3-K4 on the target promoters. RBP2 mRNA and protein expression increased in 71.5% （30/42） and 100% （20/20） of primary gastric cancer specimens, respectively. Significantly diminished foci numbers coupled with massive senescence and elevated expression of the cyclin-dependent kinase inhibitors （CDKIs） p21^CIP2, p27^kip1 and/or p16^ink4a, occurred in the RBP2 depleted gastric and cervical cancer cells. RBP2 knocking-down enhanced the promoter activity of all three CDKIs genes. RBP2 occupied these promoter regions in the control cells and the loss of RBP2 occupancy was accompanied by enhanced H3-K4 tri- methylation following RBP2 depletion.All the research showed FoxM1 and RBP2 is over-expressed in gastric cancer and their inhibition triggers senescence of malignant cells by derepressing the CDKI target genes. Cell senescence induced by FoxM1 inhibition and histone demethylase inhibition by targeting RBP2 may be the novel strategy against gastric cancer.更多还原