DLC-1基因甲基化检测与肝细胞癌复发转移的关系

目的:研究DLC-1基因甲基化检测与肝细胞癌(hepatocelluar cardnoma,HCC)复发转移的关系.方法:73例HCC标本依据临床以及病理学特征被分为高侵袭组和低侵袭组;采用甲基化特异性PCR对不同侵袭组HCC之间DLC-1基因甲基化表达进行分析.结果:DLC-1甲基化表达率高侵袭组明显高于低侵袭组,二者之间有明显差异(χ2=4.3567,P<0.05).DLC-1甲基化阳性与阴性患者之间AFP、HBV双阳性率有明显差异(χ2=4.4224,P<0.05);TNM分期越后DLC-1甲基化程度越高(χ2=10.8478,P<0.05);短期随访发现,DLC-1甲基化的HCC患者中位生存期低于非甲基化患者(9.45 mo vs 36 mo,P<0.05).结论:DLC-1基因甲基化可作为HCC复发转移监测指标,并可作为靶向治疗HCC复发转移的新靶点.     

肝细胞癌发生中p16基因缺失与甲基化的研究

目的 探讨肝细胞癌（ｈｅｐａｔｏｃｅｌｌｕｌａｒ ｃａｒｃｉｎｏｍａ，ＨＣＣ）发生过程中抑癌基因Ｐ１６的存在及蛋白表达情况。方法 随机引物法标记制备Ｄｉｇ－ｐ１６ｃＤＮＡ探针，分子杂交及免疫组化方法检测肝细胞癌发生中Ｐ１６基因的存在及表达情况。结果 斑点杂交显示Ｐ１６探针敏感率为１．０ｐｇ。５６例ＨＣＣ癌组织及２８例癌旁组织ＤＮＡ与Ｐ１６探针点杂交的阳性率分别为５５．３６％（３１／５６）、８９．２     

胃癌中抑癌基因PTEN甲基化状态的检测

胃癌发生涉及多种抑癌基因、癌基因的异常表达。PTEN作为一种广泛的抑癌基因，在多种肿瘤中存在失活现象。其失活原因除突变、缺失外，DNA异常甲基化可能是其失活的第三条途径。本研究采用甲基化特异性聚合酶链反应（MSP）检测胃癌细胞和组织中PTEN基因甲基化状态，探讨其甲基化改变特点与临床病理特征的关系，为胃癌的发生机制提供分子生物学依据。     

肝细胞癌RUNX3基因甲基化与杂合缺失的分析及其意义

目的 通过筛查肝细胞癌(HCC)RUNX3遗传学和表遗传学异常,拟明确RUNX3基因在HCC发病过程中的作用。方法 采用聚合酶链反应(PCR)单链构象多态性、杂合缺失(LOH)分析、测序以及DNA甲基化特异的PCR技术对90例HCC RUNX3基因突变、LOH及甲基化状态进行检测,对RUNX3基因缺失、甲基化结果与各临床病理参数的关系进行分析。结果 未发现突变病例;但发现3个单核苷酸多态性分别存在于外显子1和4;LOH分析表明30.6％(11/36)的病例存在LOH;54.4％(49/90)的病例存在RUNX3基因高甲基化;RUNX3 LOH与HCC门静脉癌栓、肝内转移和微血管受侵差异有显著性(x~2值分别为4.729、4.581、4.581,P值均<0.05)。结论 HCC RUNX3基因存在高频率的LOH和高甲基化;RUNX3基因的异常可能在HCC发病过程中起重要作用。     

甲状腺乳头状癌组织中TSHR基因启动子甲基化研究

目的探讨甲状腺乳头状癌组织中促甲状腺激素受体基因（Thyroid Stimulating Hormone Receptor,TSHR）基因启动子区5端CpG岛甲基化改变的特点与临床特征的关系。方法采用甲基化特异性PCR（MSP,methylation-specific PCR）方法检测TSHR基因启动子甲基化情况。结果（1）22／34例甲状腺乳头状癌组织中检测到TSHR基因启动子甲基化,9／34例甲状腺乳头状癌癌旁组织中检测到TSHR基因启动子甲基化,癌组织中TSHR基因启动子甲基化率显著增高（χ^2=10．019,P=0．002〈0．05）;（2）有淋巴结转移的甲状腺乳头状癌组织（15／18例）TSHR基因启动子的甲基化显著高于无淋巴结转移组（7／16例）（χ^2=5．812,P=0．016〈0．05）。结论TSHR基因启动子异常甲基化是甲状腺乳头状癌发展过程中的分子事件之一,可能影响了甲状腺乳头状癌细胞的摄碘的功能。     

DNA低甲基化与肝细胞癌

DNA甲基化是一种表遗传修饰,是由DNA甲基转移酶（DNMT）催化以S-腺苷甲硫氨酸（SAM）作为甲基供体将胞嘧啶转变为5-甲基胞嘧啶的一种反应,这种修饰反应通常发生在CpG二核苷酸上.CpG二核苷酸在人类基因组中约占10%,其分布是非随机的,其中70%～80%呈甲基化状态,这种甲基化修饰主要集中在CpG密度较低的区域及DNA重复序列,而另一小部分CpG密度较高的区域称为CpG岛,通常位于基因的5＇端,覆盖基因启动子及第一外显子.人类基因组中约有45000个CpG岛,这些CpG岛通常未被甲基化[1].     

原发性肝细胞癌中p16基因甲基化及其蛋白表达分析

目的探讨p16基因的甲基化改变和p16蛋白表达在肝癌的发生发展过程中的作用。材料和方法收集原发性肝细胞癌(HCC)手术切除的新鲜标本10例,石蜡包埋标本3 3例,Ⅰ级8例,Ⅱ级2 1例,Ⅲ级14例(Edmonson分级) ,p16蛋白检测用免疫组化法(ABC) ,p16基因甲基化检测用甲基化特异PCR(MS_PCR)法。结果HCCp16蛋白缺失率65 1% ( 2 8/4 3 ) ,而癌旁组织缺失率11 5 % ( 3 /2 6) ,两者相比有显著差异(P <0 0 1)。Ⅰ～Ⅱ级HCCp16蛋白的缺失率与Ⅲ级相比明显较低(P <0 0 5 )。p16蛋白阳性的15例HCC标本,均未检出基因甲基化,p16蛋白缺失的2 8例标本,有19例检出基因甲基化,甲基化率44 2 %。10例癌旁肝组织有1例基因甲基化,HCC和癌旁组织相比甲基化率有明显差异(P <0 0 5 ) ,基因甲基化与p16蛋白缺失明显相关(P <0 0 1)。各级HCCp16基因启动子区甲基化率有明显差异(P <0 0 5 )。结论p16蛋白缺失与HCC的发生有密切关系,低分化的HCC和p16蛋白缺失有关,p16蛋白在判断其恶性程度方面有一定的价值。基因的甲基化可能是p16基因在HCC中的主要失活方式,HCC分化程度和p16基因甲基化之间有密切关系。     

BRCA1启动子甲基化与肝癌的关系研究

目的探讨BRCA1基因启动子区甲基化与肝癌发生的关系。方法采用甲基化特异性PCR检测肝癌细胞系和肝癌组织标本的甲基化状态。结果5个肝癌细胞系中2个(PLC-PRF-5和97H)发现甲基化,3个未发现甲基化;在39例肝癌组织标本中22例发生甲基化(56.4%)。结论BRCA1启动子甲基化与肝癌的发生有关,可能作为肝癌的诊断标志物。     

肝细胞癌中wwox基因启动子甲基化与蛋白表达的关系

目的:探讨wwox基因启动子甲基化及蛋白表达与肝细胞性肝癌的关系.方法:通过甲基化特异性PCR(methylation specific polymerase chain reaction,MSP)方法及免疫组织化学(immunohistochemestry,IHC)法分别检测60例肝细胞性肝癌组织和癌旁组织中wwox基因启动子甲基化状态和蛋白表达水平.结果:癌组织及癌旁组织中wwox基因启动子甲基化阳性率分别为41.67%(25/60)和8.33%(5/60)(P=0.000).WWOX蛋白在癌和癌旁组织中的表达具有显著性差异[35.00%(21/60)vs70.00%(42/60),P=0.001].wwox基因启动子甲基化和蛋白表达与肝外转移、肿瘤直径、肿瘤细胞分化密切相关(P=0.007,0.014,0.011);WWOX蛋白表达与临床分期、肿瘤直径、肿瘤细胞分化密切相关(P=0.018、0.023、0.001).wwox基因启动子甲基化与蛋白表达显著负相关(γ=-0.408,P=0.001).结论:启动子区甲基化是wwox基因失活的重要机制.wwox启动子区异常甲基化可能参与了肝癌的发生发展,在肝癌的进展发挥重要作用.     

肝细胞癌中存活素mRNA的表达与凋亡、增殖和组织侵入性特征的相关性

存活素对于肝癌致病性和预测方面的临床意义已有所探讨。为了研究存活素对HCC患在肿瘤行为和预测方面的临床作用，作对40例HCC组织和对应相邻肝组织标本以及7位健康对照肝组织标本的存活素mRNA表达采用实时逆转录PCR法进行了检测。存活素mRNA的表达水平（10g拷贝数／毫微克总RNA）在健康肝组织中为1．95＋／-0．44，在癌旁有病的肝组织中为4．79＋／-0．96，而在HCC组织中为5．87＋／-0．73（P〈0．001）。     

肝细胞肝癌谷胱甘肽硫转移酶P1活性和甲基化研究

与环境中致癌物黄曲霉毒素B1（AFB1）密切接触可增加肝细胞肝癌（HCC）的危险性。进入体内的AFB1在肝内经细胞色素P4501A2活化为AFB1-8.9环氧化物（前致癌物），在肝细胞胞浆中与谷胱甘肽S-转移酶（GST）携带的活性极团谷胱苷肽结合后，水溶性增加，     

肝癌组织中侧群细胞的检测及初步分析

随着对肿瘤研究的不断深入及对干细胞了解的日益加深,越来越多的研究证实,一些肿瘤组织中存在肿瘤干细胞.由于没有特异性的表面标记分子,迄今为止尚无成熟的肝癌干细胞的分离与鉴定技术,从而导致对肝癌干细胞的研究举步维艰.侧群(SP)细胞有着和组织特异的肿瘤干细胞几乎相同的功能和分子特点,快速的Hoechst 33342染料排斥特性可以应用到多种肿瘤干细胞的分离和纯化中,为肿瘤干细胞研究提供了更为便利的途径.本研究采用Hoechst 33342染色法从肝癌患者的肝癌组织中分离、培养肝癌SP细胞,并对其进行初步分析,为进一步研究其生物学特性奠定基础.     

Patterns of methylation profiles as diagnostic markers for multiple hepatocellular carcinomas

BACKGROUND: Hepatocellular carcinoma often displays multiple tumor nodules, thus posing a problem for differential diagnosis between cancers of both multifocal and metastatic origin. Conventionally, pathological criteria have been used for this purpose, but these are largely subjective. In order to facilitate a more objective differential diagnosis of multiple HCCs, we used the patterns of methylation of p16INK4a, p14ARF, and GSTP1 genes as markers for each tumor nodule. METHODS: Sixty-seven nodules from 30 cases of multiple or recurrent HCCs were examined using methylation-specific PCR (MSP) analysis for the detection of methylation profiles. RESULTS: Hypermethylation was detected in 56.7%, 43.3% and 17.9% of the cases for p16INK4a, p14ARF, and GSTP1 genes, respectively. At the genetic level the inter-nodule methylation profiles were heterogeneous in 23 of the cases and homogeneous in another 7, enabling a multifocal origin to be diagnosed in the former and metastatic origin in the latter. CONCLUSIONS: Methylation profiling seems to be useful in differentiating the clonal origins of multiple cancers, as the information yielded by this method is essentially objective.     

Epigenetic silencing of WIF-1 in hepatocellular carcinomas

Purpose

To examine the expression profile and promoter methylation status of WIF-1 in hepatocellular carcinoma (HCC) and identify the possible relationship between the WIF-1 expression pattern and promoter methylation status.

Methods

Quantitative real-time PCR was performed to detect mRNA level of WIF-1 in 4 HCC cell lines, 15 paired HCC clinical samples and 3 normal liver tissues. Methylation-specific PCR and bisulfite DNA sequencing were used in methylation analysis. In vitro assays for HCC cells, colony formation and cell proliferation assay were carried out to observe the effect of WIF-1 on cell growth; TOP-flash luciferase analysis was employed to determine its role in the Wnt pathway.

Results

Quantitative real-time PCR analysis showed the extensive low expression of WIF-1 mRNA in HCC, and this down-regulation was generally dependent on the degree of methylation at its promoter region. In vitro assays indicated WIF-1 can inhibit cell growth by blocking Wnt signaling in HCC cells.

Conclusions

WIF-1 silencing as a result of its promoter hypermethylation may be a frequent event in HCC.     

Alterations of DNA methylation and histone modifications contribute to gene silencing in hepatocellular carcinomas

Aim: The aim of the present study was to examine DNA methylation and histone modification changes in hepatocellular carcinomas (HCC). Methods: DNA methylation in the P16, RASSF1a, progesterone receptor (PGR) and estrogen receptor alpha (ERalpha) promoters was determined by quantitative bisulfite-pyrosequencing technique in HCC patients. Histone H3-lysine (K) 4, H3-K9 and H3-K27 modifications in all these four genes were examined by chromatin immunoprecipitation (ChIP) assay in HCC cell lines. Expression of two DNA methyltransferases (DNMT1 and DNMT3b) and three histone methyltransferases (SUV39H1, G9a and EZH2) in HCC patients was measured by real-time polymerase chain reaction. Results: Aberrant DNA methylation was detected in all the HCC. Patients with DNA methylation in the RASSF1a, PGR andERalpha promoters in cancers also had substantial DNA methylation in their non-cancerous liver tissues, whereas DNA methylation in the P16 promoter was cancer specific. Epigenetic states in HCC cell lines showed that silencing of P16 and RASSF1a depended on DNA methylation and histone H3-K9 methylation. However, silencing of the PGR and ERalpha genes was more closely related to H3-K27 methylation rather than DNA methylation. Consistent with the alteration of histone status, higher expression of G9a and EZH2 was found in HCC than in non-cancerous liver tissues (P < 0.01). Conclusion: These data suggest that multiple epigenetic silencing mechanisms are inappropriately active in HCC cells.     

SOCS1基因与肝细胞癌的关系研究进展

SOCS1基因位于16p12-p13.1,它编码蛋白SOCS1并属于SOCS蛋白家族,通过抑制JAK—STAT信号转导通路发挥作用,其失活机制主要是甲基化和杂合性缺失。SOCS1在肝细胞癌中广泛甲基化和表达明显降低,提示SOCS1可能是抑癌基因,在肝细胞癌的发生发展中起十分重要的作用。     

The role of hMLH1 methylation in the development of synchronous sporadic colorectal carcinomas

PURPOSE: AB. B. subset of sporadic colorectal carcinomas show microsatellite instability, usually as a result of biallelic hMLH1 gene promoter methylation. Synchronous tumors occur in up to 5 percent of patients with colorectal cancer, but their cause is poorly understood. We hypothesized that in the setting of sporadic microsatellite instability cancers, synchronicity may reflect a global predisposition of colorectal epithelium toward tumor development because of gene hypermethylation. METHODS: We identified 14 individuals with 33 synchronous cancers from a series of 362 patients with 381 sporadic colorectal cancers. We then analyzed the synchronous lesions for microsatellite status, hMLH1 protein expression, and hMLH1 promoter methylation. RESULTS: Seven of 33 synchronous tumors (21 percent) showed microsatellite instability, compared with 36 of 348 solitary tumors (10.3 percent, P = 0.06). The 14 patients with synchronous tumors were significantly older than those with solitary tumors (mean age 79.4 vs. 68.2 years, P = 0.01), and 5 of these patients had at least one microsatellite instability tumor. However, only one patient harbored synchronous tumors that were all of the microsatellite instability type. Methylation of the hMLH1 promoter was seen in 9 synchronous cancers from 27 assessable lesions in 7 patients and was associated with microsatellite instability (P = 0.01), right-sidedness (P = 0.01), and loss of expression of hMLH1 (P = 0.03). Only one case showed methylation in all synchronous tumors, whereas in five cases synchronous tumors showed different methylation status within the one individual. CONCLUSION: Our data suggest that synchronous tumors arise as independent events and that the slightly greater frequency of synchronous tumors in individuals with microsatellite instability cancers is likely to be a chance event reflecting the older age of these individuals rather than arising from a predisposition toward cancer as a result of global hypermethylation of colorectal epithelium.