肺癌胰岛素样生长因子-2基因印迹的研究

目的探讨胰岛素样生长因子-2（IGF2）基因印迹（genomic imprinting）与肺癌发生发展过程的关系。方法于2003年1月至2004年1月，对大连医科大学附属第一医院胸外科手术切除的标本，根据IGF2基因第9外显子具有ApaI位点多肽性，利用聚合酶链反应（PCR）技术结合限制性片段长度多态性（RFLP）技术，诊断的32例肺癌患者及其对应的癌周正常肺组织进行了IGF2基因印迹的研究。结果12例患者为杂合子信息个体（37，5％），其中10例为IGF2双等位基因表达，即发生了基因印迹缺失（LOI，83．3％），而且这10例病人中有4例的癌周正常肺组织表现为IGF2弱的双等位基因表达。结论IGF2基因的印迹缺失参与了肺癌的发生发展过程。     

非小细胞肺癌MEST基因的表达及印迹状态

目的探讨中胚叶特异性转录子(又名父源表达基因-1)(MEST)的基因印迹在非小细胞肺癌发生发展中的作用。方法利用聚合酶链反应(PCR)技术结合限制性片段长度多态性(PFLP)技术,分析32例非小细胞肺癌及其对应的癌周正常肺组织中MEST基因的表达及其印迹状态。结果 11例MEST杂合子信息样本中,9例(81.8%)肺癌组织发生了印记缺失(LOI),其中6例为低等级、低发展阶段的肿瘤,而与之对应的癌周组织除1例为弱的双等位基因表达之外,均为单等位基因表达。同时11份MEST杂合子信息样本肺癌组织中MEST的平均表达水平是癌旁组织的1.5倍,且二者表达量差异有显著性(P0.01)。结论 MEST基因的印迹缺失参与了非小细胞肺癌的发生发展过程。     

人肝癌胰岛素样生长因子2基因的表达和印迹状态的改变

目的 研究人肝细胞性肝癌(HCC)的发生与胰岛素样生长因子2(IGF2)的表达及其印迹变化之间的关系。方法 采用RT-PCR半定量法和限制性酶切片断长度多态性分析法(RFLP),对40例HCC组织标本(其中33例有癌旁组织),检测IGF2的相对表达量,观察肝癌组织中IGF2基因印迹状态的改变。结果 HCC中IGF2的相对表达量,在各病例间的变化较大;癌组织中的表达(1.5431±1.4316)明显高于癌旁肝组织(0.6517±0.6666),t=3.695,P<0.001;癌组织和癌旁组织均有病例发生基因印迹丢失(LOI)。结论 HCC的发生与IGF2的异常表达增高有关;IGF2的LOI可能是HCC的癌前表现之一。     

非小细胞肺癌中IGF1、IGFBP2表达与预后的相关性分析

目的探讨非小细胞肺癌组织胰岛素样生长因子1(IGF1)、胰岛素样生长因子结合蛋白2(IGFBP2),蛋白表达与患者临床病理特征及预后的关系。方法将226例确诊的非小细胞肺癌患者癌组织为非小细胞肺癌组;选取其癌旁正常组织为癌旁对照组。检测癌组织及癌旁正常组织IGF1、IGFBP2蛋白表达水平;分析非小细胞肺癌患者癌组织IGF1与IGFBP2蛋白表达相关性及其与预后的关系;并分析影响非小细胞肺癌患者预后的因素。结果非小细胞肺癌组IGF1、IGFBP2蛋白高表达率高于癌旁对照组(P<0.05)。非小细胞肺癌患者癌组织IGF1、IGFBP2蛋白表达与肿瘤大小、淋巴结转移、组织分化程度、TNM分期有关(P<0.05)。非小细胞肺癌患者癌组织IGF1与IGFBP2蛋白表达呈正相关(r=0.472,P<0.05)。IGF1、IGFBP2高表达患者五年生存率(33.33%、29.41%)低于低表达患者(69.23%、73.33%)(P均<0.05)。IGF1高表达、IGFBP2高表达、TNMⅢ~Ⅳ期是影响非小细胞肺癌患者死亡的独立危险因素(P<0.05)。结论IGF1、IGFBP2可能在非小细胞肺癌发生发展过程中发挥重要作用,且IGF1、IGFBP2高表达与患者不良预后有关。     

人肝癌父系表达基因10的遗传印记状态

目的 研究人肝癌组织及肝癌细胞株中父系表达基因10(PEG10)的遗传印记状态.方法 从40例肝癌及其癌旁组织、15例正常肝组织、5株肝癌细胞(PLC/PRF/5、SMMC 7721、HepG2、Hep3B、SK-HEP-1)、2株正常肝细胞(changliver、HL7702)中提取基因组DNA,针对PEG10基因单核苷酸多态性位点设计引物进行PCR,扩增片段经测序分析基因型;从杂合样本中提取总RNA进行RT-PCR,对扩增产物测序以检测等位基因表达状态,同时进行实时荧光定量RT-PCR检测PEG10表达水平.计量资料以均数±标准差(-x±s)表示,组间比较用t检验与方差分析;两组率的比较用x2检验.结果 40例肝癌及其癌旁组织中16例呈杂合状态,15例正常肝组织中3例呈杂合状态,肝癌细胞HepG2扩增片段测序检测到一杂合突变位点,其余组织及细胞株均为纯合状态.杂合样本中,82.4%(14/17)肝癌样本(包括组织及肝癌细胞株)中PEG10基因呈双等位基因表达,发生印记丢失;17.6%(3/17)肝癌样本呈单等位基因表达,提示印记存在.PEG10在癌旁及正常肝组织中几乎不表达.发生印记丢失的肝癌组织与印记存在的肝癌组织相比,PEG10表达水平的差异无统计学意义(t=1.311,P＞0.05).结论 大多数肝癌组织中存在PEG10印记丢失现象,PEG 10印记状态与其在肝癌组织中的表达水平无明确关系.     

肺癌及癌旁组织中经典Wnt信号途径相关基因的甲基化状态观察

目的 观察人肺癌及癌旁组织中经典Wnt信号途径相关基因的甲基化状态,探讨其与肺癌发生的关系.方法 应用巢式甲基化特异性聚合酶链反应(nMSP)检测25例新鲜肺癌组织及癌旁组织、11例存档肺癌组织、6例炎性假瘤的瘤旁正常肺组织以及肺腺癌A549细胞株的WIF-1、sFRP-1和DKK-3基因启动子区域的甲基化状态.结果 6例正常肺组织未检测到甲基化基因.18例肺癌组织和10例癌旁组织中可检测到甲基化基因.在13例肺癌组织和2例癌旁组织可检测到2个以上甲基化基因.肺癌组织中WIF-1、sFRP-1和DKK-3三个基因的甲基化率分别为47.2%、36.1%、27.8%;肺腺癌A549细胞株中WIF-1和DKK-3基因呈甲基化状态,而8FRP-1基因呈未甲基化状态.结论 肺癌组织中存在经典Wnt信号途径相关基因(WIF-1、sFRP-1、DKK-3)的甲基化.其导致的基因功能失活可能发生在肺癌形成的早期.     

肺癌患者PTEN基因启动子甲基化和转录表达的研究

目的 探讨张力蛋白同源的磷酸酶基因(PTEN)启动子CpG岛甲基化状态和基因表达水平与肺癌发生的关系.方法 用甲基化特异性PCR检测PTEN启动子CpG岛甲基化状态,实时定量PCR 检测PTEN mRNA的表达水平.结果 在肺癌组织中PTEN基因启动子甲基化的频率为26.67%(12/45),在肺正常组织中未发生甲基化(χ2=7.448,P＜0.01).正常组织中PTEN mRNA全部表达,肺癌组织中表达缺失率为22.22%(10/45),且表达量低于正常肺组织(t=-14.156,P＜0.001),不同年龄、性别、肿瘤大小、恶性程度、肿瘤分类中其表达量无显著性差异(P＞0.05);PTEN甲基化与其mRNA表达下降密切相关.结论 肺癌中PTEN基因启动子甲基化频率明显升高,其表达普遍下调或缺失,提示PTEN启动子甲基可在肺癌发生、发展中起一定作用.     

FHIT基因在肺癌中的缺失与HPV感染相关性的研究

目的　探讨肺癌发生的分子生物学机制 ;方法　采用逆转录 -巢式聚合酶链反应 (reverse- tape polymerase chainreaction)的方法对 4 2例肺癌及 10例正常肺组织中的 FHIT(Fragile histidine triad)基因的缺失情况进行检测 ,并用 PCR技术检测了肺癌组织中人乳头状瘤病毒 (human papilloma virus,HPV)的 DNA片段 ;结果　 6 6 .7% (2 8/ 4 3)肺癌组织中检测到 FHIT基因的缺失 ,而正常组织中未检测到 FHIT基因的缺失 ,二者差别有显著意义 (p<0 .0 1)。 4 2例肺癌组织中有 8例检测到 HPV的片段 ,阳性率为 19% (8/ 4 2 ) ,正常组织中未检测到 HPV的片段 ,且在 8例 HPV阳性的标本中均有 FHIT基因缺失。结论　 FHIT基因的缺失在肺癌的发生中起一定的作用 ;可与肺癌的不同病理分型、分化程度及临床分期无关 ;但与吸烟有一定的相关性 ;同时 HPV的感染与肺癌的发生有一定关系 ,且与 FHIT基因缺失呈正相关     

NORE1A基因在肺癌组织中的表达缺失及相关因素分析

目的探讨抑癌基因NORE1A在肺癌组织中的表达缺失情况及其临床意义。方法采用RT-PCR技术检测46例肺癌组织及相应癌旁正常组织中NORE1A基因的表达缺失情况。结果(1)NORE1A基因在正常肺组织均有表达,在肺癌组织中表达缺失率为45%;(2)NORE1A基因在不同病理细胞类型标本中的表达缺失率不存在明显差异,P>0.05;(3)吸烟组和非吸烟组间NORE1A基因的表达缺失比较差异无显著性,吸烟组缺失率为48%,非吸烟组为42%(P>0.05);(4)淋巴结转移组的NORE1A基因的表达缺失率高于无淋巴结转移组标本,分别为60%和18%(P<0.01)。结论不能认为吸烟是导致NORE1A基因失活的一个具有重要影响的因素。NORE1A基因的表达缺失与肺癌发生有关,不仅发生于早期肺癌,而且可能在其晚期亦存在继发性大量失活。     

p73基因蛋白表达与老年人肺癌临床病理学特征及预后关系的探讨

目的探讨p73基因蛋白与老年人肺癌发生、发展的关系。方法应用免疫组化SABC法检测了65例老年人肺癌组织中抑癌基因p73基因蛋白的表达,并与癌旁组织和正常肺组织对比,同时结合肺癌的临床病理学特征及预后进行分析。结果老年人肺癌组织中p73基因蛋白阳性表达率(477%)明显高于癌旁组织(92%)和正常肺组织(46%),P<005;不同年龄组、不同类型和不同分化程度肺癌组织中p73基因蛋白的阳性表达率差异无显著性(P>005);Ⅰ期和Ⅱ期老年人肺癌组织中p73基因蛋白阳性表达率(605%)明显高于Ⅲ期和Ⅳ期(227%),P<005;生存3年或以上的老年人肺癌组织中p73基因蛋白阳性表达率(722%)明显高于生存3年以下者(160%),P<005。结论老年人与非老年人肺癌在p73基因蛋白免疫组化染色表达中有共同性,与正常肺组织或癌旁组织相比明显增加,对于进一步探讨p73基因蛋白在肺癌发生发展中的表达及调节有参考价值;p73基因蛋白表达在各型肺癌发生、发展中可能具有普遍意义,可作为判断老年人肺癌生物学行为和预后的一个参考指标。     

Epigenetic changes encompassing the IGF2/H19 locus associated with relaxation of IGF2 imprinting and silencing of H19 in Wilms tumor.

In most tissues IGF2 is expressed from the paternal allele while H19 is expressed from the maternal allele. We have previously shown that in some Wilms tumors the maternal IGF2 imprint is relaxed such that the gene is expressed biallelically. We have now investigated this subset of tumors further and found that biallelic expression of IGF2 was associated with undetectable or very low levels of H19 expression. The relaxation of IGF2 imprinting in Wilms tumors also involved a concomitant reversal in the patterns of DNA methylation of the maternally inherited IGF2 and H19 alleles. Furthermore, the only specific methylation changes that occurred in tumors with relaxation of IGF2 imprinting were solely restricted to the maternal IGF2 and H19 alleles. These data suggest that there has been an acquisition of a paternal epigenotype in these tumors as the result of a pathologic disruption in the normal imprinting of the IGF2 and H19 genes.     

Monoallelic expression and methylation of imprinted genes in human and mouse embryonic germ cell lineages

Imprinting is an epigenetic modification leading to monoallelic expression of some genes, and disrupted imprinting is believed to be a barrier to human stem cell transplantation, based on studies that suggest that epigenetic marks are unstable in mouse embryonic germ (EG) and embryonic stem (ES) cells. However, stem cell imprinting has not previously been examined directly in humans. We found that three imprinted genes, TSSC5, H19, and SNRPN, show monoallelic expression in in vitro differentiated human EG-derived cells, and a fourth gene, IGF2, shows partially relaxed imprinting at a ratio from 4:1 to 5:1, comparable to that found in normal somatic cells. In addition, we found normal methylation of an imprinting control region (ICR) that regulates H19 and IGF2 imprinting, suggesting that imprinting may not be a significant epigenetic barrier to human EG cell transplantation. Finally, we were able to construct an in vitro mouse model of genomic imprinting, by generating EG cells from 8.5-day embryos of an interspecific cross, in which undifferentiated cells show biallelic expression and acquire preferential parental allele expression after differentiation. This model should allow experimental manipulation of epigenetic modifications of cultured EG cells that may not be possible in human stem cell studies.     

Loss of genomic imprinting of insulin-like growth factor 2 is strongly associated with cellular proliferation in normal hematopoietic cells

OBJECTIVE: The human insulin-like growth factor 2 (IGF2) gene was thought to be imprinted and expressed only from the paternal allele in normal tissue. MATERIALS AND METHODS: Initially, we analyzed the imprinting status of IGF2 in bone marrow cells from 49 patients with myelodysplastic syndromes (MDS) utilizing the Apa I polymorphism of IGF2. Thirteen bone marrow and 14 peripheral blood samples from normal individuals served as controls. We utilized normal peripheral blood T lymphocytes to examine the relationship between genomic imprinting and cell proliferation. Expression of IGF2 was quantified by real-time PCR and proliferation of T cells was measured by 3H-thymidine incorporation. Furthermore, methylation status of the imprinting controlling region (ICR) was analyzed by subcloning and sequencing of genomic DNA after sodium bisulfite modification. RESULTS: Among 24 patients who were heterozygous for IGF2, loss of imprinting (LOI) occurred in 22 cases (92%). Surprisingly, LOI of IGF2 occurred in the normal bone marrow cells, but the normal peripheral blood cells showed retention of imprinting (ROI). Unstimulated normal T cells showed ROI. After 24 hours of exposure to PHA, these cells changed their IGF2 imprinting status from ROI to LOI. Concomitantly, their IGF2 RNA levels increased up to sixfold and their proliferation increased 10- to 20-fold. In contrast, normal T cells not stimulated with PHA did not develop LOI of IGF2, had negligible levels of IGF2 RNA, and did not increase their proliferation. In unstimulated T cells, the CpG islands of the ICR were completely methylated on one allele and nearly completely unmethylated on the other allele. After PHA stimulation, the CpG islands at the ICR became completely methylated on both alleles. CONCLUSION: LOI of IGF2 is strongly associated with cell proliferation and is not limited to cancer cells.     

Loss of imprinting of insulin growth factor II gene: a potential heritable biomarker for colon neoplasia predisposition

BACKGROUND & AIMS: Loss of genomic imprinting (LOI) of insulin-like growth factor II gene (IGF2) involves abnormal activation of the normally silent maternally inherited allele. LOI of IGF2 has been associated with personal and family history of colorectal neoplasia (CRN), supporting a role for LOI in colorectal carcinogenesis. Whether LOI of IGF2 is associated with known environmental risk factors for CRN is unknown. METHODS: We performed quantitative hot-stop PCR for imprinting analysis of IGF2 on normal peripheral blood lymphocytes (PBL) of individuals. Environmental exposures including tobacco, alcohol, NSAIDs, and nutrient consumption (calcium, folate, selenium, fiber, and fat) were correlated with LOI expression in PBL. Odds ratios (OR) and 95% CI were calculated. RESULTS: The prevalence of LOI of IGF2 was examined in 172 individuals. Persons with CRN (adenomas/cancer) had 5.1-fold (95% CI: 1.92-13.6) increased risk of having LOI of IGF2 in PBL compared with those without CRN. In contrast, tobacco smoking (OR = 0.96, 95% CI: 0.36-2.55), alcohol consumption (OR = 1.22, 95% CI: 0.45-3.3), and NSAIDs use (OR = 1.21, 95% CI: 0.38-3.94) were not significantly associated with LOI of IGF2. Nutrient ingestion including calcium (P = 0.61), folate (P = 0.23), selenium (P = 0.19), fiber (P = 0.63), and fat (P = 0.14) was not statistically correlated with LOI of IGF2. CONCLUSIONS: Abnormal imprinting of IGF2 gene was strongly associated with CRN but not with any of the environmental exposures examined. LOI of IGF2 does not appear to be an environmentally acquired phenomenon but rather a hereditary risk factor for CRN.     

Loss of imprinting of the insulin-like growth factor II gene occurs by biallelic methylation in a core region of H19-associated CTCF-binding sites in colorectal cancer

We hypothesize that loss of imprinting (LOI) of the insulin-like growth factor II (IGF2) gene is associated with a predisposition to sporadic colorectal cancer. We confirmed a previously known strong correlation between LOI and microsatellite instability and showed that LOI was not a consequence of microsatellite instability or mismatch repair deficiency. LOI of IGF2 correlated strongly with biallelic hypermethylation of a core of five CpG sites in the insulator region of IGF2/H19, which is a known CTCF-binding element. As this methylation-dependent LOI was present in both tumors and normal colonic mucosa, it is possible that hypermethylation creates a field defect predisposing to cancer.