人肝癌细胞遗传印记基因PEG10印记状态不受DMR甲基化调控

目的探讨人肝癌细胞遗传印记基因(genetic imprinted gene)PEG10差异性甲基化区(differntially DNA methylated region,DMR)甲基化状态在其印记调控中的作用。方法以单核苷酸多态性(single nucleotide polymorphisms,SNP)位点为等位基因标记,分析人肝癌HepG2细胞PEG10印记状态;采用RT-PCR、免疫组化及Western-blot检测PEG10表达水平;重亚硫酸盐修饰DNA测序法分析PEG10-DMR甲基化状态;再应用甲基基团供体S-腺苷蛋氨酸(S-adenosyl-L-methionine,SAM)及DNA甲基转移酶抑制剂5-氮杂胞苷(5-azacytidine,5-azaC)在体外及荷瘤裸鼠体内调控HepG2细胞PEG10-DMR甲基化状态,观察PEG10-DMR甲基化状态改变对PEG10印记状态及表达水平的影响。结果遗传印记基因PEG10在HepG2细胞呈双等位基因表达的印记丢失。与正常肝细胞HL7702相比,其PEG10-DMR甲基化水平显著升高。调控PEG10-DMR甲基化水平可改变PEG10的表达水平,但对其印记状态无影响。结论 PEG10-DMR甲基化不参与人肝癌细胞PEG10的印记调控,但可调控其表达水平。     

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。     

雄激素和雄激素受体对肝癌细胞株PEG10表达的作用

目的 探讨雄激素和雄激素受体(AR)对肝癌细胞株PEG10表达的调控作用.方法 设计合成针对人ARsiRNA,并转染HepG2和7404肝癌细胞株.用双氢睾丸酮(DHT)干预HepG2细胞.Western Blot检测AR和PEG10表达水平.结果 从3对AR siRNA中筛选到1对siRNA(AR siRNA-3),它在2种肝癌细胞株中均可有效抑制AR的表达,其抑制作用呈剂量依赖关系.2种肝癌细胞株中,浓度为240 nmol/L的AR siRNA-3在转染后24 h,对AR抑制效率可达80%以上,且抑制效果可持续至72 h.AR siRNA-3转染24h后PEG10表达水平降低,转染48 h后,PEG10表达水平降低非常明显,72 h后PEG10表达有所上升.DHT可促进HepG2细胞PEG10的表达,呈剂量依赖关系.DHT对AR表达未见明显作用.结论 雄激素和AR参与了肝癌细胞株PEG10表达的调控.这可能是男性肝细胞癌发病率较高的原因之一.     

人遗传印记基因PEG10对L02细胞线粒体膜电位的影响

人遗传印记基因PEG10定位于人7号染色体长臂2区1带,是一个反转座子衍生而来的父系表达的遗传印记基因.PEG10基因高表达于肝癌细胞系HepG2和大多数肝细胞癌组织中,在良性肝病组织及非肝脏来源的肿瘤细胞系中没有检测出该基因的表达.PEG10基因的表达水平与肝细胞癌的转移潜能正相关,并且可以促进正常人肝细胞L02增殖,提示PEGl0基因可能在人肝细胞癌的发生过程中起重要作用.本研究通过检测L02细胞转染前后线粒体膜电位及增殖细胞核抗原(PCNA)表达的改变,初步探讨PEG10基因促人肝细胞增殖的机制.     

遗传印记基因PEG10在结肠癌中的表达及意义

目的研究遗传印记基因PEG10在结肠癌、癌旁组织、正常结肠组织中的表达及意义。方法收集中国人民解放军第一七四医院2015年1月-2015年9月40例结肠癌、癌旁组织,20例正常结肠组织标本,通过免疫组织化学及蛋白印迹法检测PEG10的细胞定位及其表达差异。结果 PEG10细胞主要分布于细胞质,在结肠癌中呈过表达,其表达明显高于癌旁组织、正常结肠组织,差异有统计学意义(P0.01)。结论 PEG10可能参与结肠癌发生、发展,但其具体作用机制仍需进一步探讨。     

MiR-122调控肝癌遗传印记基因PEG10的实验研究

目的 探讨遗传印记基因PEG10功能的异常与miRNA失调控的相关性.方法 通过生物信息学网站TargetScan预测可能参与PEG10调控的miRNA分子,筛选到miR-122.通过基于taqman探针的实时定量逆转录聚合酶链反应,比较miR-122在原代正常肝细胞和3株肝癌细胞株(Huh7,Hep3B,HepG2)中的表达差异.将miR-122的分子前体转染HepG2细胞,观察转染前后PEG10在mRNA和蛋白水平的表达变化.多组均数问比较采用秩和检验,配对样本组间差异比较采用t检验.结果 生物信息学预测结果显示,miR-122可能参与了PEG10的调控.实时定量逆转录聚合酶链反应结果显示,PHHC、Huh7,HepG2、Hep3B细胞miR-122的表达量(2~(-△△Ct)值)分别为1.0578±0.0975、0.5600±0.0632、0.0068±0.0012、0.0058±0.0008,H=9.667,P＜0.05.与原代正常肝细胞相比,miR-122在肝癌细胞株中表现为完全(Hep3B、HepG2细胞)或部分缺失(Huh7细胞),其表达水平与PEG10呈负相关.将miR-122分子前体转入HepG2细胞后,PEG10在mRNA水平并未显示出明显的下调,但Western blot结果提示miR-122分子前体明显抑制了PEG10蛋白的表达.结论 miR-122参与了遗传印记基因PEG10的调控,其调控主要发生在翻译阶段,即蛋白质水平.miRNA的失调控与肝癌的发生密切相关,其功能的异常可能早于受其调控的癌基因或抑癌基因的改变,是肝癌发生的早期事件,这为肝癌的早期预警和基因治疗提供了新思路.     

PEG10在人胃癌组织中的表达及其临床意义

目的研究印记基因10(paternally expressed gene 10,PEG10)在胃癌组织中的表达情况,并探讨PEG10与胃癌的临床病理特征及预后的关系。方法利用免疫组化技术检测84例胃癌患者癌组织、癌旁组织及10例正常胃组织中PEG10表达情况,并结合其临床资料分析其临床意义。结果 84例胃癌组织中有63例(75.00%)PEG10阳性表达,相应癌旁组织中有50例(59.52%)PEG10阳性表达,差异有统计学意义(χ~2=4.601,P=0.032),而10例正常胃组织PEG10均阴性表达。PEG10的阳性表达与淋巴结转移(χ~2=6.115,P=0.013)及肿瘤的TNM分期相关(χ~2=7.710,P=0.007)。Cox多因素回归分析表明,PEG10阳性表达是患者预后不良的独立危险因素(P=0.03)。结论 PEG10在胃癌组织中高表达,在癌旁组织中相对低表达,而在正常胃组织中不表达。PEG10的表达与淋巴结转移及TNM分期相关。PEG10对胃癌患者预后判断有一定的价值,可能成为一个新的胃癌分子标志物。     

肝癌基因谱动态表达及HSPgp96异常的临床价值

目的:探讨肝细胞癌变过程中基因表达谱的动态变化, 观察热休克蛋白(HSP)gp96表达及其在肝癌早期诊断及判断预后方面的临床价值. 方法:以2-乙酰氨基芴(2-FAA, 0.05%)喂饲SD大鼠诱发肝癌发生, 观察肝病理组织学(HE染色)改变, 用全基因组芯片分析肝基因表达谱的动态变化, 以巢式逆转录酶链反应扩增gp96基因片段.结果: SD大鼠在喂饲2-FAA后, 肝细胞在诱癌早期发生颗粒样变性, 中期出现不典型增生, 后期见大量癌巢结节. 肝癌发生过程中大鼠肝基因表达谱明显改变, 与对照大鼠比较, 癌变大鼠gp96上调3倍; 诱癌变过程中, 肝组织gp96 mRNA表达明显增加, 变性组, 癌前组和癌变组的肝组织中gp96基因表达均明显高于正常对照组(90.9%, 100%, 100% vs 16.67%, P<0.05). 结论:肝细胞癌变过程中基因表达谱明显改变, 其中gp96 mRNA呈过度表达, 其分析有利于肝癌的诊断和预后判断.     

肝癌组织中一氧化氮合酶及其基因表达的研究

目的研究肝癌组织中一氧化氮合酶(iNOS)及其基因表达与肝癌发生发展的关系。方法用免疫组化和原位杂交的方法对21例肝癌及癌旁组织中的诱导型一氯化氮合酶(iNOS)及其基因表达进行原位检测和观察。结果:NOS 阳性反应物质呈黄色或棕黄色,位于细胞浆中。非癌殖织(肉眼观距癌组织边缘>1.5)多呈阴性或弥漫弱阳性,但部分非癌组织中可见 iNOS 呈阳性的细胞呈点状分布;癌旁组织多呈阳性,提示 iNOS 表达与肝组织癌变有关。癌组织核心多呈阴性或弥漫弱阳性,但分化中和差的癌组织核心也分别有一例 NOS 呈强阳性;周边癌组织呈局灶阳性,侵入纤维组织中的弥敢癌细胞星强阳性,提示 NOS 的表达与肝癌组织的侵润能力有关。肝癌组织 iNOSmRNA 阳性细胞的分布与 iN-OS 蛋白的表达基本相似。结论 iNOS 蛋白及其基因表达与肝组织癌变及肝癌侵润能力有关。     

肝癌相关基因表达的荧光定量PCR检测及分子诊断指数的建立

目的:研究肝细胞癌相关基因表达,建立肝癌分子诊断指数,以期能更准确的诊断肝癌.方法:采用实时荧光定量PCR检测40例肝细胞癌患者癌组织和配对的癌旁2 cm及手术切缘组织、10例肝硬化组织、10例正常肝脏组织中11个基因的表达,以管家基因G3PDH为对照,2-ΔΔCT法计算目的基因相对表达量,挑选出特异性好且与正常肝、肝...     

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.     

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。     

肝癌细胞对5-氮杂-2'-脱氧胞苷的敏感性与细胞总DNA甲基化水平无关

目的 比较不同肝癌细胞株对5-氮杂-2'-脱氧胞苷(5-aza-dC)的敏感性,探讨肝癌细胞对5-aza-dC的敏感性是否与细胞总DNA甲基化水平有关.方法 用不同剂量(0.5、5.0、10.0μmol/L)的5-aza-dC处理肝癌细胞株(HepG2、QGY7701和HepG2.2.15细胞)及正常肝细胞株L02,比较不同浓度处理前后的细胞增殖抑制率,比较10 μmol/L 5-aza-dC处理前后的Caspase-3活性及细胞DNA片段化水平(5-溴脱氧尿嘧啶核苷掺入率),比较不同细胞总DNA甲基化水平.组间检测结果比较采用t检验.结果 5-aza-dC对HepG2、QGY7701、HepG2.2.15、L02细胞的半数抑制浓度分别为0.5、0.5、4.5、11.4μmol/L,与HepG2细胞和QGY7701细胞相比,HepG2.2.15绌胞和L02细胞对5-aza-dC不敏感.HepG2和QGY7701细胞中Caspase-3的活性升高较L02和HepG2.2.15细胞明显(P值均＜0.05),QGY7701细胞中5-溴脱氧尿嘧啶核苷掺入率升高较L02细胞明显(P＜0.05).L02、HepG2、QGY7701和HepG 2.2.15细胞的DNA总甲基化水平分别为11.7%±0.9%、10.9%±1.3%、11.7%±1.7%和12.2%±1.0%,差异无统计学意义(P值均＞0.05).结论 细胞对5-aza-dC的敏感性与细胞总DNA甲基化水平无关.     

GABA stimulates human hepatocellular carcinoma growth through overexpressed GABAA receptor theta subunit

AIM: To investigate the function of gamma-aminobutyric acid (GABA) and gamma-aminobutyric acid A receptor θ subunit (GABRQ) in hepatocellular carcinoma (HCC).METHODS: Semiquantitative polymerase chain reaction was used for detecting the expression of GABRQ receptor among HCC cell line HepG2, normal liver cell line L-02, non-malignant Chang’s liver cells, 8 samples of HCC tissues and paired non-cancerous tissues. HepG2 cells were treated with GABA at serial concentrations (0, 1, 10, 20, 40 and 60 μmol/L), and their proliferating abilities were analyzed with the methyl thiazolyl tetrazolium assay, cell cycle analysis and tumor implanted in nude mice. Small interfering RNA was used for knocking down the endogenous GABRQ in HepG2. Proliferating abilities of these cells treated with or without GABA were analyzed.RESULTS: We identified the overexpression of GABRQ in HCC cell lines and half of the tested HCC tissues. Knockdown of endogenous GABRQ expression in HepG2 attenuated HCC cell growth, suggesting its role in HCC cell viability. We studied the effect of GABA in the proliferation of GABRQ-positive cell lines in vitro and in vivo, and found that GABA increased HCC growth in a dose-dependent manner. Notably, the addition of GABA into the cell culture medium promoted the proliferation of GABRQ-expressing HepG2 cells, but not GABRQ-knockdown HepG2 cells, which means that GABA stimulates HepG2 cell growth through GABRQ.CONCLUSION: GABRQ play important roles in HCC development and progression and could be a promising molecular target for the development of new diagnostic and therapeutic strategies of HCC.     

Autophagy related protein 9A increase in hepatitis B virus-associated hepatocellular carcinoma and the role in apoptosis

The majority of hepatocellular carcinoma (HCC) cases are associated with the hepatitis B virus (HBV) infection. Autophagy related protein 9A (ATG9A) is a transmembrane protein required for autophagosome formation. In order to investigate the role of ATG9A in HBV-associated HCC, ATG9A protein expression was determined in tumor liver tissues and compared with adjacent nontumor tissues from HCC patients with or without HBV infection. In HBV-associated HCC tissues, ATG9A protein level was increased in tumor liver tissues, but not in cases of non-HBV HCC. Our findings suggested that ATG9A might be involved in HBV and cancer cell survival. Therefore, we aimed to analyze the function of ATG9A in HBV replication using RNA interference to evaluate the HBV DNA level using real-time PCR. In the present study, there were no significant differences between shATG9A-transfected HepG2.2.15 cells and the mock control. However, we found that silencing ATG9A affected apoptosis in HepG2.2.15 and HepG2 cell lines. Our results indicated that ATG9A might be partly involved in the survival of HCC. Thus, the inhibition of ATG9A together with other targets might be a potential drug target for HCC treatment.     

Decaprenyl diphosphate synthase subunit 2 as a prognosis factor in hepatocellular carcinoma

AIM:To investigate the involvement of decaprenyl diphosphate synthase subunit 2(PDSS2) in development and progression of human hepatocellular carcinoma(HCC).METHODS:PDSS2 protein expression was examined in well-and poorly differentiated HCC tumor samples.The levels of PDSS2 expression were compared with clinical features and prognosis of HCC patients.The effects of PDSS2 on cell proliferation,cell cycle,apoptosis,cell migration,and invasion in HCC Hep G2 cells were also investigated.RESULTS:PDSS2 was downregulated in poorly differentiated cancer samples compared with welldifferentiated tumor samples,and the expression level was markedly lower in HCC tissues than in histologically normal tissue adjacent to the cancer.Reduced protein expression was negatively associated with the status of HCC progression.In addition,overexpression of PDSS2dramatically suppressed cell proliferation and colony formation,and induced apoptosis in Hep G2 cells by inducing G1-phase cell-cycle arrest.The migration and invasion capabilities of Hep G2 cells were significantly decreased following PDSS2 overexpression.CONCLUSION:Decreased PDSS2 expression is an unfavorable prognostic factor for HCC,and PDSS2 has potent anticancer activity in HCC tissues and Hep G2cells.     

Detection of eukaryotic translation initiation factor 4E and its clinical significance in hepatocellular carcinoma

AIM: To study the expression of eukaryotic translation initiation factor 4E (eIF4E), which is closely correlated with malignant tumors, and its relationship to prognosis in hepatocellular carcinoma.METHODS: Western blotting was performed to quantify the elF4E protein expression in the normal human liver cell line L02 and the hepatoma cell lines Hep3B, HepG2, and Huh7. Forty-six hepatocellular carcinoma samples with complete clinical data were obtained from Changzheng Hospital during the period of December 2008 to July 2009. The expression of eIF4E in the tumor samples and their adjacent tissues were detected by immunohistochemistry. The relationship between the test results and hepatocellular carcinoma (HCC) prognosis was statistically analysed by using a COX proportional hazard model.RESULTS: Western blotting analysis showed that there were distinct eIF4E protein bands in all three of the hepatoma cell lines. In particular, the HepG2 cell line had the highest level of eIF4E protein expression. The L02 cell group had a low eIF4E expression. Immunohistochemical assay showed that there were 32 cases in which the tumour tissue expression was higher than their adjacent tissues, accounting for 69.57%. There were also 14 cases in which the tumour tissue expression was lower or no significant difference was found, accounting for 30.43%. COX proportional hazards model analysis showed that HCC prognosis was related to the depth of invasion, the overexpression of eIF4E and p53, possibly as independent HCC prognostic predictors.CONCLUSION: In summary, eIF4E expression is associated with liver cancer, and patients with high eIF4E expression levels have a higher risk of recurrence.     

Differential expression of hDAB2IPA and hDAB2IPB in normal tissues and promoter methylation of hDAB2IPA in hepatocellular carcinoma

BACKGROUND/AIMS: hDAB2IP is a candidate tumor suppressor gene. We studied the expression of its two variants, hDAB2IPA and hDAB2IPB, in normal tissues, and the expression and methylation status of hDAB2IPA in hepatocellular carcinomas (HCC) and cell lines. METHODS: Conventional or real-time RT-PCR was performed in normal tissue samples, cell lines and HCC samples, and sequencing analysis and methylation-specific PCR in cell lines and HCC samples. RESULTS: hDAB2IPA was the predominant isoform, being expressed in the majority of tissues examined. The expression of hDAB2IPA was silenced or down-regulated but could be restored by 5-aza-2'-deoxycytidine treatment in liver cancer cell lines. The reactivation of hDAB2IPA was associated with promoter demethylation. The correlation between promoter methylation and hDAB2IPA expression was confirmed in eight pairs of matched HCC samples. Further, the methylation of the hDAB2IPA promoter in HCC was confirmed in an additional 53 pairs of patient samples. More than 80% of HCC samples showed hDAB2IPA promoter methylation, compared to 11.5% in the corresponding adjacent normal tissue (p<0.0001, chi2). CONCLUSIONS: Our data suggest that hDAB2IPA is the dominant isoform expressed in normal tissues. Its expression is suppressed in HCC, consistent with its role as a tumor suppressor gene, mainly by promoter methylation.