【作者】 张琼；

【导师】 林菊生；

【作者基本信息】 华中科技大学 ， 内科学， 2007， 博士

【摘要】 研究背景与目的原发性肝细胞癌(hepatocellular carcinoma;HCC)是一种全球性高度恶性的肿瘤。根据2002年WHO的统计,全世界HCC发病率占男性肿瘤的第4位,女性肿瘤的第7位。每年近50万人死于本病。在我国,在过去短短的20年中,HCC从恶性肿瘤死因的第3位跃居第2位。肝癌的临床特点是:肝癌早期无特殊症状,病情隐匿,不易被发现,70%-80%患者确诊时已属临床晚期(III或IV期)。肝细胞癌具有易复发和转移性,这也是导致癌症患者治疗效果不佳和死亡的最重要、最常见的原因。尽管近年来涌现出许多新的治疗手段,但HCC的预后未得到明显改善。肿瘤的基因治疗是近年来研究的热点,发现了许多与肝癌发病相关的靶分子,如p53、p16、c-myc、PTEN等,但研究表明,它们都只在部分肝癌中起致癌作用且缺乏高度的特异性。因此,寻找参与绝大多数肝癌发病的关键性的特异分子靶是现阶段国内外分子肿瘤学的研究热点。PEG10(paternally expressed gene 10)是2001年Ryuichi Ono等用cDNA微阵列在肝细胞癌组织中发现的一个新的遗传印记基因(genetic imprinting gene),来源于病毒反转录转座子,位于人类染色体7q21上。所谓遗传印记,又称基因组印记,是指根据遗传信息的亲代来源决定等位基因的一个表达,而另一个“沉默”,即等单位表达。遗传印记基因影响哺乳动物细胞的生长、发育和行为,与人类先天性疾病、发育不全和肿瘤等密切相关。Tsou AP和Okabe H等进一步研究发现:PEG10在小鼠再生肝和人类肝细胞癌组织中高表达,在相应的癌旁及正常肝组织中不表达。检测PEG10的氨基酸序列发现也有一个CCHC型的锌指结构。因而推测PEG10也可能参与转录调控。而且这种结构在大部分逆转录病毒和一些逆转座子中也可以找到。最感兴趣的是我们首次发现:PEG10基因在人消化道其他肿瘤细胞系中弱表达,而在多种肝癌细胞系中高表达;且PEG10的表达量与肝癌细胞系的转移潜能成正相关,故此我们选择PEG10为分子靶,初步评价PEG10的表达与肿瘤恶性表型、转移等的相关性。因此本课题对PEG10这个分子靶标的功能进行深入研究。基于其在肝细胞癌和原发性肝癌组织中的特异高表达性,通过基因沉默或者遗传修饰等手段控制PEG10的功能或它的调节通路,将是一个令人振奋的分子治疗靶点。方法荧光实时定量PCR和RT-PCR检测五种不同转移潜能的肝癌细胞系中PEG10基因mRNA的表达水平;在肝癌细胞系HepG2中提取RNA,扩增PEG10表达全长;利用真核表达载体pcDNA3.1hisC构建重组质粒后,转染人正常肝细胞系LO2中并通过G418筛选出稳定表达PEG10的LO2细胞株,命名为LO2-PEG10;应用RT-PCR、Western Blot检测稳定转染细胞株中PEG10的表达水平;应用荧光定量PCR检测过表达PEG10基因的LO2细胞中p16、p21基因mRNA的表达变化,应用Western Blot法检测p16、p21、CyclinD1、CyclinE、CDK4、CDK2、Rb、pRB(ser780)、E2F1的表达变化;采用MTT、TUNEL、CFSE-流式细胞术和体外细胞迁移实验、Transwell体外侵袭实验、细胞黏附实验、荧光示踪等方法观察PEG10基因对正常肝细胞株LO2的细胞增殖、运动、体外侵袭和黏附能力的影响;激光共聚焦显微镜成像观察PEG10表达对LO2细胞骨架的影响;透射电镜观察LO2-PEG10细胞的超微结构变化;体外克隆平板实验检测LO2-PEG10细胞体外成瘤能力;肝癌裸鼠模型实验观察PEG10过表达对肝癌侵袭转移表型的影响。结果1.应用荧光定量PCR分析三株不同转移潜能的肝癌细胞系中PEG10基因mRNA量的表达,首次发现PEG10基因的表达量和肝癌转移潜能呈正相关。2.成功构建了含PEG10基因全长的真核表达载体,获得稳定表达PEG10的LO2细胞克隆株及其对照人肾细胞株293。3.将重组载体转染不表达PEG10基因的正常肝细胞株LO2,结果发现促进细胞增殖,促进细胞从G1期向S期转换,肿瘤细胞周期缩短,细胞凋亡受到明显抑制。p16、p21蛋白表达下调,而CyclinD1、CyclinE、CDK4、CDK2、Rb、pRB(ser780)、E2F1蛋白表达上调,这提示我们PEG10可能是通过作用于肝细胞的细胞周期和抑制细胞凋亡而发挥致癌作用的。4.过表达外源性PEG10,通过改变细胞极性和超微组织结构,促进人肝细胞LO2具有侵袭和转移的恶性生物学表型。5.动物体内试验表明, PEG10基因使人肝细胞LO2具有成瘤性,并具有向其它器官侵袭和转移能力。结论采用荧光定量PCR技术在不同转移潜能肝癌细胞株中检测PEG10基因mRNA表达量,应用分子克隆技术构建重组真核表达载体,转染正常肝细胞株LO2和非肝脏来源的人肾细胞株293,筛选出稳定表达PEG10基因全长的LO2和293细胞株,并以PEG10基因为靶点作进一步研究。验证了PEG10基因的表达量与肝癌的转移潜能成正相关。过表达PEG10的LO2细胞株通过p16、p21二条抑癌基因信号通路改变下游通路细胞周期蛋白的表达,具有恶性生物学表型:增殖、侵袭和转移的能力;而过表达PEG10对非肝脏来源的人肾细胞株293没有影响。进一步探讨PEG10基因参与肝癌侵袭转移的分子机制发现:外源性PEG10基因下调E-cadherin、β-catenin胞内信号,促进人肝细胞增殖、迁移和成瘤能力;改变E-cadherin、β-catenin维持细胞极性和组织结构的功能,促进人肝细胞LO2的转移表型。动物体内实验也证实PEG10过表达增强肝癌增殖,促进肝癌发生肝内转移和远处转移及腹腔种植转移。综上所述,PEG10基因可能是通过细胞周期介导肝细胞发生恶性生物学表型,下调E-cadherin、β-catenin胞内信号促进肝细胞侵袭转移。这也提示我们PEG10基因将是治疗肝癌的一个有效的分子靶标。更多还原

【Abstract】 Backgroud & Aims: HCC is a global highly malignant tumor. According to the WHO statistics in 2002, the incidence of HCC ranks 4th position in male human tumors and 7th position in female human tumors. Each year nearly 50 million people died of the disease. In China, over the past two decades, HCC ranks 2nd from 3rd among the cause of death, Clinical characteristics of early stage HCC is a symptomatic, occult and difficult to detect. 70% - 80% patients are already at the advance stage (III or IV) when they are go to see their doctors. HCC is very often characterized by recurence and metastasis, which contribute to the poor outcome and high matality. Notwithstanding a variety of novel therapeutics strategies for HCC emerged in recent years, the prognosis of HCC has not been noticeably improved. Nowadays gene therapy for cancers tumors has become the hot topics, molecular target relevant to HCC, such as p53、p16、c-myc、PTEN etc were explored, but the study shows that they play roles only in the part of tumors and lack high specificity. Hunting for specific molecular target which play an important role in the development of majority of HCC is the hot interests in the field of molelar oncology at home and abroad.PEG10, paternally expressed gene 10, a new genetic imprinting gene, was found by Ryuichi in 2001 in hepatocellular carcinoma tissue by means of cDNA microarray, originated from retrotranscription transposon, is located on human chromosome 7q21. So-called genetic imprinting, also named genome imprinting, refers to the genetic information according to parental origin to determine the expression of allele, while another‘silent’. Genetic imprinting genes affect mammalian cell growth, development and behavior, and closely relate to human genetic diseases, hypoplasia and tumors. Tsou Ap and Okabe H is results that PEG10 was highly expressed in the regenerative liver of mice and in human HCC tissue but not surrouding adjacent tissue and normal hepatic tissue. PEG10 amino acid sequence was detected to contain a CCHC type zinc finger which can be found in most retrovirus and retrotransponsons. Therefore it can be inferred that PEG10 may be involved in transcriptional regulation. Most interesting is the first discovery that PEG10 was lightly expressed in human hepatoma cell line and other cancer cell line derived from gastrointestinal carcinoma, and the expressive quantity of PEG10 has the positive correlation with the metastatic potential. In view of the high specificity of the expression of PEG10 in HCC tissue and hepatoma cell lines, PEG10 was chosen as the molecular target for the evaluation of the relationship between the expression of PEG10 and malignant phenotype and metastasis of the tumor. In this study, PEG10 as a molecular target was investigated intensively and extensively by using gene silencing and genetic modification. As expected, the results obtained were very exciting: PEG10 may be a potential promising molecular target for gene therapy of HCC.Methods: the mRNA of PEG10 was examined in different HCC lineages by real-time quantitative fluorescence PCR and RT-PCR. Total RNA was extracted in hepatoma cell line HepG2 and was amplified the full-length of PEG10 by RT-PCR; Then we recombinant the plasmid with the eukaryotic expression vector pcDNA3.1hisC, normal liver cell line LO2 was transfected PEG10 and screened the clone cells use of G418; The PEG10 gene’s mRNA and protein expression level were detected in LO2-PEG10 cells by RT-PCR and Western blot; the expression change of p16, p21 gene in LO2 cells by fluorescence quantitative PCR, The protein expression of p16, p21, Cyclin D1, Cyclin E, CDK4, CDK2, Rb, pRB (ser780) and E2F1 were detected by Western blot; the proliferate and adhere affection, migrate and invasive ability of PEG10 gene in LO2 cells were measured by MTT、TUNEL、CFSE flow cytometry、Transwell tests etc. Laser scanning confocal microscope imaging cytoskeleton in LO2 cells which over expressed PEG10; At the same time the changes of cells ultrastructure in LO2-PEG10 cells was observed by TEM; and the ability of tumor-growing in vitro was measured with soft agar colony formation. nude mice model observation PEG10 expression of liver cancer invasion and metastasis phenotypic effects Finally we also evaluated the invasion and metastasis influence on over expression due to PEG10 in use of HCC nude mice model.Results:1. The expression of PEG10 mRNA in five HCC lineages with different metastasis potential was detected by FQ-PCR, we found that the expression levels of PEG10 gene in liver cancer cells were positively correlated with their different metastatic potentials.2. PEG10 stable expression of the LO2 cell clone and the control cell line 293. Recombinant plasmid contains the full length PEG10 was constructed successfully. LO2 cells which treated with PEG10 was stable screened, at the same time human renal cell line 293 was used as control group.3. The recombinant vector was stably transfected into the normal liver cells LO2. It was found that exogenous PEG10 promotes cell proliferation which changes the acceleration from G1 to S phase and inhabits cells apoptosis. The expression of P16, p21 protein was down-regulated, however, the expression of Cyclin D1, Cyclin E, CDK4, CDK2, Rb, pRB (ser780), E2F1 protein was up-regulated. Doubtless the above findings clearly suggests us PEG10 maybe educe carcinogenic action by influencing the hepatic cells cell cycle.4. Exogenous PEG10 assign human liver cells LO2 the phenotype of invasion andmetastasis by changing cell polarity and ultrastructure.5. Animal experiments showed that exogenous PEG10 makes the LO2 cells heve the ability of tumor-growing in vivo and promotes cells invasion and migration. Conclusions: PEG10 mRNA expression was detected in different metastatic potential hepatoma cell lines by Real-time PCR technology analysis, recombinant plasmid was constructed with molecular cloning technology, normal liver cells LO2 and the non-liver derived cells 293 were stably transfected PEG10 gene, screening cells which stably expressed PEG10gene, and take PEG10 gene as a target for further study.By way of a series of experiments, the expression level of PEG10 was verified that it occurs positive correlation to the metastasis ability of Hepatoma cell lines. The LO2-PEG10 cells changes the cell cycle protein expression through p16, p21 two tumor suppressor gene signal pathways downstream, PEG10 gene makes LO2 cells have malignant phenotype: proliferation, invasion and metastasis, however, the non-liver-derived cell line 293 will not be affected.During the time in studying the molecular mechanism how PEG10 to participate in HCC, it was found that exogenous PEG10 gene can down regulated E-cadherin/catenin intracellular signal, promote human liver cells LO2 proliferation, movement and tumor growing in vitro; change the ability of E-cadherin/catenin complex to maintain cell polarity and ultrastructural functions, assign human liver cells LO2 the metastatic phenotype. It was testify doubtless that the over expression of PEG10 can promote the following abilities of tumor-growing of HCC cells in vivo and metastasis to other organs.From all the above, we may reach the conclusion that PEG10 gene change the malignant biological phenotype of LO2 cells and down-regulating the cells signal pathway. Therefore, it also indicates that PEG10 will be an effective molecular target for liver cancer gene therapy.更多还原