【作者】 谭晓虹；

【导师】 韦长元；

【作者基本信息】 广西医科大学 ， 肿瘤学， 2010， 博士

【摘要】 肝细胞性肝癌(hepatocellular carcinoma,HCC,以下简称肝癌)是全世界最常见的恶性肿瘤之一,有着较高的发病率和死亡率。我国是世界上肝癌的高发地区之一,每年新发HCC病例占全世界新发病例数的比例高达42.5%,严重威胁着人民的身体健康。手术切除和肝脏移植是目前治疗肝癌较为有效的方法,但由于肝癌具有恶性程度高、发展迅速、容易复发和转移等特点,肝癌患者中仅有一部分人适合外科手术治疗。绝大部分肝癌患者只能依赖于放疗、化疗及其它的一些姑息疗法。因此深入研究肝癌的病因及发病机理,对于寻找有效的肝癌化学预防及治疗方法无疑具有十分积极的意义。肝癌的发生和发展是一个多因素、多步骤、多基因作用的复杂过程,研究表明,p21(p21WAF1/CIP1)可能通过不同的途径参与了肝癌的发生和发展。p21属于细胞周期抑制因子(CKIs)中的Cip/Kip家族,可以通过抑制细胞周期蛋白/细胞周期依赖性激酶(cyclin/CDK)复合物活性,使细胞周期进程受阻;而且还可以通过抑制细胞增殖核抗原(PCNA)与DNA聚合酶δ(polδ)结合,从而抑制DNA合成。现已确定人的p125亚基是由POLD1基因编码的, POLD1表达受到细胞周期的调控,但其机制尚未明了。癌基因的活化、抑癌基因的失活或突变以及激素调节、信号转导和转录调控等的失调均可作用于细胞周期调控系统而激活细胞进行不正常的DNA复制,最终出现细胞的异常增殖导致肿瘤的发生。鉴于p21在细胞周期调控和DNA复制中所起的重要作用,本研究通过真核表达载体使p21高表达和慢病毒载体靶向沉默p21表达,研究p21表达量变化对肝癌细胞SMMC-7721生物学行为的影响,双向验证了p21对细胞周期进程的抑制作用,在肝癌侵袭转移中可能具有的作用,并初步探讨了p21对POLD1基因表达的影响,从而为下一步深入研究肝癌细胞中p21对POLD1基因的表达调控通路打下了坚实的基础。目的:通过真核表达载体使p21高表达和慢病毒载体靶向沉默p21表达对肝癌细胞SMMC-7721生物学行为的影响,验证p21对细胞周期进程的影响,在肝癌侵袭转移中是否发挥作用,并初步探讨在肝癌细胞中p21对POLD1基因表达的影响。方法:1.瞬时转染:将表达p21 cDNA的真核表达载体pXJ41-p21及空载体pXJ41-neo转染入SMMC-7721细胞;将p21小干扰RNA片段p21-siRNA、阴性对照片段NC转染入SMMC-7721细胞。2.稳定转染:SMMC-7721细胞瞬时转染pXJ41-p21及空载体pXJ41-neo后,经G418筛选,获得稳定细胞系7721-p21、7721-pXJ;用包装有p21小干扰RNA片段p21-siRNA及阴性对照片段NC的慢病毒载体感染SMMC-7721细胞,经有限稀释法获得稳定细胞系7721-p21RNAi、7721-NC。3. RT-PCR、Western-blot检测瞬时转染及稳定转染后p21、POLD1的mRNA和蛋白表达水平。4.通过生长曲线测定,流式细胞仪检测细胞周期和凋亡,克隆形成实验,侵袭和迁移能力测定,了解SMMC-7721细胞在p21表达水平改变后生物学行为的变化。结果:1.瞬时转染p21 48h后发现,p21 mRNA表达水平升高, POLD1 mRNA表达水平降低,并且SMMC-7721细胞生长速率下降,G0/G1期占细胞周期比例升高,S期比例下降(P<0.05)。对高表达p21的稳定细胞株研究发现,随着p21 mRNA及蛋白表达水平升高, POLD1 mRNA及蛋白表达水平随之降低;细胞生长受抑制,G0/G1期比例升高,细胞凋亡比例及克隆形成数减少,迁移及侵袭能力减弱。2.瞬时转染p21小干扰RNA片段48h后发现,p21 mRNA表达水平降低, POLD1 mRNA表达水平升高,并且SMMC-7721细胞生长速率升高,G0/G1期占细胞周期比例下降,S期比例升高(P<0.05)。对p21低表达的细胞株研究发现,随着p21 mRNA及蛋白表达水平下降, POLD1 mRNA及蛋白表达水平随之增强;细胞生长速度加快,G0/G1期比例下降,S期比例升高,细胞凋亡比例及克隆形成数增多,迁移及侵袭能力增强。结论:p21可以抑制SMMC-7721细胞的细胞周期进程,对抗凋亡的发生,有可能参与了肝癌的侵袭转移的发生。POLD1基因的表达水平与p21呈负相关,p21可能参与了POLD1基因的调控,并且这种作用有可能是p21对细胞增殖抑制及细胞恶性表型变化的调控途径之一。更多还原

【Abstract】 Hepatocellular Carcinoma(HCC) is one of the most common carcinomas,contributes to the higher morbility and mortality among cancer patients because of its highest malignancy.HCC endangers the lives and health of Chinese people because each year the newly developed hepatic cancer patients in the Chinese mainland accout for 42.5 percent of the the cases in the world.In perspective,surgical therapy and liver transplantation are effective,but very few patients are indicative of surgical operations due to high malignancy,fast growth,susceptibility for relapse and metastasis and a great portion of them are tentatively treated with chemotherapy,radiotherapy as well as other alleviative treatments.Therefore, a thorough alternative understanding of the pathogenesis of HCC thus holds the promise of finding an effective chemoprevention and treatment for this cancer.The formation of HCC is a multi-factor, multi-step and multi-gene process.The studies on the pathogenesis of HCC shows that p21 has involved in the genesis and development of HCC. p21(p21WAF1/CIP1)belongs to the Cip/Kip family of CDK inhibitors. The p21 gene encodes a 21kDa protein which binds and inhibits cyclin/CDK complex , predominantly blocks G1/S phase cell cycle transition. p21 has also been shown to inhibit PCNA binding to DNA polymerase delta (polδ)and hereby block DNA replication. polδis a cell cycle-dependent protein with important roles in DNA replication,repair, recombination and cell cycle regulation. DNA polymerase delta is encoded by the POLD1 gene, the transcription of which is strictly cell cycle-dependent, but the mechanisms has not yet well known.The replication of eukaryotic chromosomes is a complex but highly regulated process. If DNA replication is perturbed, abnormally replicated DNA may lead to tumorigenesis.As the profound role of p21 in cell cycle regulation and DNA replication ,our research studied the behavior effects on HCC cell line SMMC-7721 through regulating the expression of p21.We confirmed the role of p21 in cell cycle arrest ,the probable influence on the invasion and metastases of HCC,and the association with POLD1gene.Objective: To study the behavior effects on SMMC-7721 cells through regulating the expression of p21.To confirm the role of p21 in cell cycle arrest ,the probable influence on the invasion and metastasis of HCC,and the association with POLD1gene.Methods:1. Transient transfection:SMMC-7721 cells were transient transfected by p21 eukaryotic expression vector named pXJ41-p21 and negative control vector named pXJ41-neo. The chemically synthesized small interfering RNA(siRNA) targeted on p21were transient transfected into cells. 2. Construction of stable cell lines: the stable cell line with p21 overexpression was obtained after G418 selection. The stable cell line with p21 knocked-down was obtained by limiting dilution assay after infected by lentivirus-mediated target silenced p21. 3.The expression of p21 and POLD1 mRNA was detected by RT-PCR. The expression of p21 and POLD1 protein was detected by Western-blot. 4.The behavior effects of cells were explored by cell growth curve, cell clones formation, transwell migration and invasion assay,flow cell cytometry. Results:1.When cells were transient transfected with p21,the levels of p21 mRNA were up-regulated at 48h,and the levels of POLD1 mRNA were down-regulated.Cell proliferation was significantly inhibited, the number of cells blocked in G0/G1 increased (P<0.05).The levels of p21 mRNA and protein were higher in the cells stably transfected with p21, accompanied with down-regulation of POLD1 mRNA and protein expression.The abilities about cell proliferation ,invasion and migration were inhibited, cell cycle progression was blocked in G0/G1 phase and apoptosis rate was lower in comparison with control groups.2. When cells were transient transfected with siRNA targeted on p21,the levels of p21 mRNA were down-regulated at 48h,and the levels of POLD1 mRNA were up-regulated.Cell proliferation was significantly increased,cell cycle arrest in G0/G1 phase was inhibited and apoptosis rate was higher.The levels of p21 mRNA and protein were down-regulated in the stable cell line knocked down p21, accompanied with up-regulation of POLD1 mRNA and protein expression.The abilities about cell proliferation ,invasion and migration were significantly increased, cell cycle arrest in G0/G1 phase was inhibited and apoptosis rate was higher.Conclusion:The results demonstrate that p21 gene can induce G0/G1 phase arrest and inhibit the cell cycle progression in SMMC-7721 cells.p21 also can protect cells from apoptosis,and involve in the invasion and metastasis of HCC probably.The expression of POLD1 correlates negatively with p21 expression,and p21 probably involves in regulation of POLD1.更多还原