转录抑制因子ZHX2对AFP启动子的抑制作用

目的：探讨转录抑制因子ZHX2对人甲胎蛋白（AFP）启动子的抑制作用。方法PCR扩增人ZHX2基因，构建ZHX2与绿色荧光蛋白和HA-tag融合表达载体pEGFP-ZHX2、pcDNA-ZHX2-HA，共转染后通过荧光显微镜及Western blot检测融合基因的表达。扩增人AFP核心启动子269bp插入pGl3-Basic，构建报告质粒phAF269。将pcDNA-ZHX2-HA和phAF269共转染HepG2细胞。48h后裂解细胞，双荧光检测分析ZHX2对AFP启动子的抑制作用。结果荧光显微镜及westem blot检测证实pEGFP-ZHX2和pcDNA-ZHX2-HA可在体外有效表达ZHX2融合蛋白，双荧光实验表明报告质粒phAF269具有AFP启动子活性，且pcDNA-ZHX2-HA与phAF269共转染HepG2后可显著抑制AFP启动子的转录作用，此抑制作用具有剂量依赖性。结论：成功构建两种新型转录抑制因子ZHX2融合表达载体，并证实其对人AFP启动子的抑制作用，为进一步探讨肝癌发生中AFP表达调控机制及提高AFP介导的靶向肿瘤基因治疗提供基础资料。     

肝细胞癌中ZHX2、NF-YA及AFP表达的相关关系

目的 检测肝细胞癌(hepatocellular carcinoma,HCC)组织中ZHX2、NF-YA及AFP mRNA和蛋白表达的相关关系.方法 采用RT-PCR及免疫组织化学方法检测HCC组织中ZHX2、NF-YA及AFP mRNA和蛋白的表达情况.结果 ZHX2 mRNA在AFP mRNA阴性的25例HCC组织中的表达率为52.0%,明显高于AFP mRNA阳性组织中的表达率(26.3%,P=0.038),ZHX2 mRNA与AFP mRNA表达呈负相关(OR=0.33);NF-YA mRNA与ZHX2 mRNA表达呈正相关(OR=31.429),而与AFP mRNA表达呈负相关关系(OR=0.308).AFP蛋白阴性的142例HCC组织中,ZHX2蛋白的表达率为23.9%,明显高于在AFP蛋白阳性组织中的表达率(12.8%,P=0.034);HCC组织中ZHX2蛋白与AFP蛋白表达呈负相关(P=0.018,r=-0.153).NF-YA蛋白在HCC组织中与ZHX2蛋白表达呈正相关(P=0.000,r=0.371),与AFP蛋白表达呈负相关(P=0.000,r=-0.497).结论 联合检测AFP和ZHX2蛋白将提高HCC的诊断率;ZHX2可能是通过NF-YA来调控AFP的表达.     

高糖诱导人肾小球系膜细胞CTGF启动子去甲基化及基因表达

目的:观察高糖刺激和甲基化抑制剂5-氮杂-2-脱氧胞苷(5-aza-dCyd)对人肾小球系膜细胞(HMCs)结缔组织生长因子(CTGF)基因启动子的甲基化水平和基因表达的影响.方法:用不同终浓度的5-aza-dCyd(0、1、2、5、10 μmol/L)刺激HMCs,于48h提取细胞基因组DNA、总RNA和蛋白质;用不同浓度的葡萄糖(5 mmol/L、30 mmol/L)刺激细胞,于0、24、48、72 h收集细胞;应用甲基化特异性PCR(MSP)检测细胞CTGF基因启动子甲基化状态,RT-PCR检测CTGFmRNA表达,Western blot检测 CTGF 蛋白表达.结果:不同浓度5-aza-dCyd处理HMCs 48 h后,0、1、2、5 μmol/L 5-azadcyd组CTGF基因启动子呈半甲基化状态,均有微量CTGFmRNA和蛋白的表达,差异无统计学意义(均P＞0.05);10 μmol/L 5-aza-dCyd组甲基化条带阴性,呈完全去甲基化状态,CTGF mRNA和蛋白表达均增加,与0 μmol/L组比较差异有统计学意义(P＜0.01).5 mmol/L葡萄糖组HMCs各时间点CTGF基因启动子均呈半甲基化状态,表达微量CTGFmRNA和蛋白质,差异无统计学意义(均P＞0.05);30 mmol/L葡萄糖组CTGF启动子在0 h呈半甲基化状态,甲基化条带较强;24 h甲基化条带减弱,48 h甲基化条带阴性,呈完全去甲基化状态,24 h、48 h、72 h CTGF mRNA和蛋白质表达均增加,与5 mmol/L组相应时间点比较差异有统计学意义(均P＜0.05).结论:高糖和甲基化抑制剂5-aza-dCyd均可诱导人肾小球系膜细胞CTGF基因启动子的去甲基化并增加CTGF的表达,提示DNA甲基化可能参与了人肾小球系膜细胞CTGF基因表达的调控.     

5-杂氮-2'脱氧胞苷对肝癌细胞SMMC-7721 miR-1247-5p基因表达及其启动子区甲基化的影响

目的观察正常人肝细胞系LO_2和肝癌细胞系SMMC-7721中miR-1247-5p的表达,研究去甲基化药物5-杂氮-2'脱氧胞苷(5-Aza-CdR)对肝癌细胞系SMMC-7721中miR-1247-5p表达及其基因启动子区Cp G岛甲基化水平的影响。方法用不同浓度(0、5和10μmol/L)去甲基化药物5-杂氮-2'脱氧胞苷处理SMMC-7721细胞,用甲基化特异性PCR法检测miR-1247-5p基因启动子区甲基化水平;用SYBR Green qReal Time PCR法检测miR-1247-5p的表达。结果与正常人肝细胞系LO_2相比,肝癌细胞系SMMC-7721中miR-1247-5p表达降低(P0.05)且其基因启动子区Cp G岛甲基化水平高;经去甲基化药物干预后miR-1247-5p的表达较对照组有明显上调(P0.01),且其基因启动子区Cp G岛甲基化水平降低。结论 miR-1247-5p基因甲基化调控可能参与了肝癌的发生。     

人肝细胞癌金属蛋白酶组织抑制因子-3的表达及与其基因启动子甲基化的关系

目的 探讨肝细胞癌的发生和门脉浸润机制。方法 20例肝细胞癌患者,每例在手术后分别取原发瘤、门脉瘤栓及远离肝癌之肝组织。用Western印迹法检测金属蛋白酶组织抑制因子(TIMP)的蛋白表达,用逆转录-聚合酶链反应(RT-PCR)检测TIMP-3 mRNA的表达,用甲基化特异性PCR检测TIMP-3基因启动子的甲基化。结果 远离肝癌之肝组织中均可见TIMP-3蛋白和mRNA的表达,原发瘤和门脉瘤栓组织TIMP-3蛋白和mRNA表达明显降低,其中分别有5例和6例完全丢失。远离肝癌之肝组织均未发现TIMP-3启动子甲基化。原发瘤中有7例、门脉瘤栓组织中有9例出现甲基化。所有有甲基化的肝癌组织,包括原发瘤和门脉瘤栓,有13例TIMP-3 mRNA和蛋白表达完全丢失,6例表达降低。TIMP-3启动子甲基化和肝细胞癌组织学分级无关(P>0.05)。结论 肝细胞癌的发生和门脉浸润与TIMP-3基因和蛋白缺失或降低相关、而TIMP-3启动子甲基化是其基因和蛋白缺失或降低的原因。     

5-aza-2dC诱导白血病细胞分化及对Annexin A1/A2表达和甲基化状态的影响

目的：观察甲基化转移酶抑制剂5-杂氮-2’-脱氧胞苷(5-aza-2’-deoxycitydine, 5-aza-2dC)对人急性髓系白血病细胞系HL-60细胞分化及对膜联蛋白A1/A2(Annexin A1/A2)表达和甲基化状态的影响。 方法：瑞氏染色和流式细胞术检测5-aza-2dC对HL-60细胞分化的影响；RT-PCR法检测药物处理HL-60细胞前后Annexin A1和A2基因mRNA的表达水平；甲基化特异性PCR(methylation-specific PCR，MSP)检测药物处理HL-60细胞前后Annexin A1和A2基因启动子区域CpG岛的甲基化水平。 结果：5-aza-2dC处理后HL-60细胞的髓系分化抗原CD11b的表达增强，细胞向成熟分化，且在0.5 μmol/L时其促分化作用最明显；Annexin A1和A2基因在HL-60细胞中低表达，0.5 μmol/L 5-aza-2dC处理HL-60细胞72 h后，Annexin A1和A2基因mRNA表达水平明显上调，而其启动子区域CpG岛甲基化水平明显降低。结论：5-aza-2dC具有促进白血病细胞分化的作用，Annexin A1和A2基因启动子去甲基化可能与5-aza-2dC诱导白血病细胞分化有关。     

DAPPER1对食管鳞状细胞癌细胞恶性生物学行为的影响及其表观遗传学调节机制

目的检测DAPPER1蛋白对食管鳞状细胞癌(esophageal squamous cell cancer,ESCC)细胞株恶性生物学行为的影响,并进一步分析ESCC组织中DAPPER1蛋白表达及引起其表达异常的可能机制。方法应用MTT、克隆形成、划痕修复实验检测DAPPER1对ESCC细胞株恶性生物学行为的影响;应用RT-PCR及甲基化特异性PCR(methylation specific PCR,MSP)技术检测细胞株(TE13、T.Tn、Eca109)中DAPPER1 mRNA的表达及其启动子区甲基化状态;应用免疫组织化学(immunohistochemistry,IHC)法检测ESCC组织中DAPPER1蛋白的表达。结果 DAPPER1在3株细胞中呈弱表达或阴性,应用甲基化抑制剂5-氮杂-2'-脱氧胞苷(5-aza-2'-deoxycytidine,5-Aza-Dc)处理细胞后,其表达强度在各细胞株中均有不同程度的增加;同时,DAPPER1过表达及5-Aza-Dc处理可明显抑制TE13细胞的增殖及迁移能力;而应用组蛋白去乙酰化酶抑制剂trichostatin A(TSA)处理细胞株后,DAPPER1在各细胞株中的表达无明显改变;DAPPER1蛋白在ESCC组织中的表达较癌旁组织明显下调(P0.01),且其表达与该基因启动子区域异常甲基化状态有关(P0.01)。结论 ESCC中DAPPER1主要起抑癌基因作用,并且该基因启动子区域的异常高甲基化可能是引起其表达下调的主要机制之一。     

小鼠神经母细胞瘤N2a细胞脑啡肽酶基因的表达与启动子区甲基化和组蛋白修饰相关

目的研究小鼠神经母细胞瘤Neuro-2a(N2a)细胞中脑啡肽酶(NEP)基因表达的表观调控机制,探讨DNA甲基化与组蛋白乙酰化之间的相互作用。方法体外培养N2a细胞,以3μmol/L、5μmol/L DNA甲基化酶抑制5-氮杂脱氧胞苷(5-Aza-dc)及(300、500、700)nmol/L组蛋白去乙酰化酶抑制剂曲古抑菌素A(TSA)分别处理N2a细胞48 h和24 h。采用逆转录PCR(RT-PCR)、Western blot法分别检测NEP的mRNA、蛋白表达;亚硫酸氢盐测序聚合酶链反应(BSP)法检测NEP基因启动子区DNA的甲基化水平;染色质免疫共沉淀(ChIP)法检测NEP基因启动子区组蛋白H3的乙酰化水平。结果 5-Aza-dc和TSA均能剂量依赖地提高NEP基因的表达(P0.01);5-Aza-dc可诱导NEP基因去甲基化(P0.01);TSA可增高NEP组蛋白H3乙酰化水平(P0.05),但不能明显改变NEP基因DNA的甲基化水平(P0.05)。结论在小鼠神经母细胞瘤N2a细胞中NEP基因的表达受DNA甲基化及组蛋白乙酰化的调控,组蛋白乙酰化水平并不影响DNA甲基化状态。     

5-氮杂胞苷对结肠癌细胞CHD5基因表达及细胞增殖活性的影响

目的研究甲基化转移酶抑制剂5-氮杂-2-脱氧胞苷(5-氮杂胞苷,AZA)对人肿瘤相关基因CHD5(染色质解旋酶DNA结合蛋白5)在结肠癌细胞中的基因转录的诱导作用以及其对细胞增殖的影响。方法 5μmol/L 5-氮杂胞苷分别作用于Lovo和SW480细胞,连续作用72 h后,用荧光定量PCR(qPCR)检测两种结肠癌细胞系中CHD5mRNA的表达,重亚硫酸盐测序(BSP)法分析启动子区的甲基化状况,MTT法分析5-氮杂胞苷对Lovo和SW480细胞增殖的影响。结果 5μmol/L 5-氮杂胞苷处理Lovo和SW480细胞72 h后,Lovo和SW480细胞中CHD5基因的发生了去甲基化,其mRNA重新表达,细胞生长受到抑制。结论 5-氮杂胞苷能够反转CHD5基因的甲基化状态,调控CHD5 mRNA表达,并能有效地抑制结肠癌细胞的增殖。     

乙型肝炎病毒X蛋白通过降低P4启动子甲基化水平上调人IGF-II基因的转录

目的:构建稳定表达乙型肝炎病毒X蛋白(HBx)的肝癌细胞株HepG2-HBx,探讨HBx对胰岛素样生长因子 II(IGF-II)基因P4启动子甲基化水平及转录表达的影响。方法:应用基因重组技术,构建含HBx基因的重组逆转录病毒载体pBABE-puro-HBx,采用磷酸钙共沉淀法将其转染293FT包装细胞产生逆转录病毒,感染HepG2肝癌细胞,采用嘌呤霉素进行阳性克隆筛选,Western blotting鉴定表达HBx蛋白的肝癌细胞株HepG2-HBx。采用亚硫酸氢盐测序法及实时荧光定量RT-PCR检测HepG2-HBx细胞中P4启动子甲基化水平及P4 mRNA表达水平变化。进一步将体外甲基化的人IGF-II基因P4启动子驱动的荧光素酶报告载体pGL3-P4及含HBx基因的pCMV-tag2B-X质粒共转染HepG2肝癌细胞,采用亚硫酸氢盐测序法及双萤光素酶实验检测pGL3-P4载体上P4启动子甲基化水平及转录调控活性变化。结果:(1)经Western blotting鉴定,成功构建了稳定表达HBx蛋白的肝癌细胞株HepG2-HBx;(2)表达HBx蛋白的HepG2-HBx细胞中P4启动子甲基化CpG位点的比例(9.0%)明显低于对照细胞HepG2-control(25.0%)(P<0.01),而其P4 mRNA表达水平则为对照细胞HepG2-control的2.8倍;(3)共转染pCMV-tag2B-X质粒的HepG2细胞中pGL3-P4载体上P4启动子甲基化CpG位点的比例(60.8%)明显低于共转染对照质粒pCMV-tag2B的HepG2细胞(84.1%)(P<0.01),而前者P4启动子相对萤光素酶活性(14.12±0.89)则明显高于后者(4.61±0.76)(P<0.01)。结论:HBx蛋白可降低IGF-II基因P4启动子甲基化水平,进而上调其转录表达。     

Cancer gene therapy by adenovirus-mediated gene transfer

Cancer arises as a direct result of genetic mutations. It therefore stands to reason that cancer should be well suited for the correction through gene therapy. Recent advances in the understanding of the molecular pathogenesis of cancer and the rapid development of recombinant DNA technology have made cancer gene therapy feasible in the clinical setting. The current efforts for cancer gene therapy mainly focus on immunogene therapy, chemogene therapy, restoration of tumor suppressor gene function, and oncolytic virus therapy. Central to all these therapies is the development of efficient vectors for gene delivery--this remains a work in progress. These vectors can be classified as viral and non-viral vectors. This paper will concentrate on viral vectors because of their practical advantages over non-viral vectors. Of the viral vectors, by far the most important are the human adenoviruses as is reflected by the enormous data and literature accumulated by studies relating to animal tumor models and clinical trials. In this review, we examine the recent progress in adenovirus-mediated cancer gene therapy with regard to cytokine gene, tumor suppressor gene, chemogene, and oncolytic adenovirus. We also discuss the current limitations of the adenoviral vector system and how they may be circumvented in future developments relating to targeted gene delivery.     

Regulatable gene expression systems for gene therapy

It is feasible to restrict transgene expression to a tissue or region in need of therapy by using promoters that respond to focusable physical stimuli. The most extensively investigated promoters of this type are radiation-inducible promoters and heat shock protein gene promoters that can be activated by directed, transient heat. Temporal regulation of transgenes can be achieved by various two- or three-component gene switches that are triggered by an appropriate small molecule inducer. The most commonly considered gene switches that are reviewed herein are based on small molecule-responsive transactivators derived from bacterial tetracycline repressor, insect or mammalian steroid receptors, or mammalian FKBP12/FRAP. A new generation of gene switches combines a heat shock protein gene promoter and a small molecule-responsive gene switch and can provide for both spatial and temporal regulation of transgene activity.     

Cancer gene therapy through autonomous parvovirus--mediated gene transfer

Parvoviruses are small nuclear replicating DNA viruses. The rodent parvoviruses are usually weakly pathogenic in adult animals, bind to cell surface receptors which are fairly ubiquitously expressed on cells, and do not appear to integrate into host chromosomes during either lytic or persistent infection. The closely related rodent parvoviruses MVM, H-1 and LuIII efficiently infect human cell lines. Most interesting, malignant transformation of human and rodent cells was often found to correlate with a greater susceptibility to parvovirus-induced killing (oncolysis) and with an increase in the cellular capacity for amplifying and / or expressing the incoming parvoviral DNA. These and other interesting properties make these autonomous rodent parvoviruses and recombinant derivatives promising candidate antitumor vectors. Capsid replacement vectors have been produced from MVM or H-1 virus that carry transgenes encoding either therapeutic products (cytokines/chemokines, Apoptin, herpes simplex virus thymidine kinase) or marker proteins (green fluorescent protein, chloramphenicolacetyl transferase, luciferase). This review describes the current state of the art regarding the potential application of wild-type parvoviruses and derived vectors for the treatment of cancer. In particular, recent successes with the development of replication-competent virus-free vector stocks are discussed and results from pre-clinical studies using recombinant parvoviruses transducing various cytokines/chemokines are presented.     

Adenoviral vector-mediated gene transfer for human gene therapy

Human gene therapy promises to change the practice of medicine by treating the causes of disease rather than the symptoms. Since the first clinical trial made its debut ten years ago, there are over 400 approved protocols in the United States alone, most of which have failed to show convincing data of clinical efficacy. This setback is largely due to the lack of efficient and adequate gene transfer vehicles. With the recent progress in elucidating the molecular mechanisms of human diseases and the imminent arrival of the post genomic era, there are increasing numbers of therapeutic genes or targets that are available for gene therapy. Therefore, the urgency and need for efficacious gene therapies are greater than ever. Clearly, the current fundamental obstacle is to develop delivery vectors that exhibit high efficacy and specificity of gene transfer. Recombinant adenoviruses have provided a versatile system for gene expression studies and therapeutic applications. Of late, there has been a remarkable increase in adenoviral vector-based clinical trials. Recent endeavors in the development of recombinant adenoviral vectors have focused on modification of virus tropism, accommodation of larger genes, increase in stability and control of transgene expression, and down-modulation of host immune responses. These modifications and continued improvements in adenoviral vectors will provide a great opportunity for human gene therapy to live up to its enormous potential in the second decade.     

胱硫醚合酶基因及其在人群中的多态性

胱硫醚合酶的活性降低或丧失是心脑血管系统等系统疾病独立的危险因子或易感因素.目前研究发现使酶活性降低或丧失的胱硫醚合酶基因的变异在人群中存在多态性.     

Genome-wide gene–gene interaction analysis for next-generation sequencing

The critical barrier in interaction analysis for next-generation sequencing (NGS) data is that the traditional pairwise interaction analysis that is suitable for common variants is difficult to apply to rare variants because of their prohibitive computational time, large number of tests and low power. The great challenges for successful detection of interactions with NGS data are (1) the demands in the paradigm of changes in interaction analysis; (2) severe multiple testing; and (3) heavy computations. To meet these challenges, we shift the paradigm of interaction analysis between two SNPs to interaction analysis between two genomic regions. In other words, we take a gene as a unit of analysis and use functional data analysis techniques as dimensional reduction tools to develop a novel statistic to collectively test interaction between all possible pairs of SNPs within two genome regions. By intensive simulations, we demonstrate that the functional logistic regression for interaction analysis has the correct type 1 error rates and higher power to detect interaction than the currently used methods. The proposed method was applied to a coronary artery disease dataset from the Wellcome Trust Case Control Consortium (WTCCC) study and the Framingham Heart Study (FHS) dataset, and the early-onset myocardial infarction (EOMI) exome sequence datasets with European origin from the NHLBI''s Exome Sequencing Project. We discovered that 6 of 27 pairs of significantly interacted genes in the FHS were replicated in the independent WTCCC study and 24 pairs of significantly interacted genes after applying Bonferroni correction in the EOMI study.     

Nuclear inheritance of a gene affecting mitochondrial gene expression

Due to a deficiency in mitochondrial protein synthesis, Chinese hamster lung (CHL) cell mutant Gal⁻ 32 does not grow in galactose or fructose. This report examines the nuclear or cytoplasmic inheritance of this single, recessive mutation. In a control experiment, fusion of Gal⁺TG^sTK⁻ cells with Gal⁻32TG^RTK⁺ cells resulted in tetraploid hybrids (as verified by karyotyping) that were selected in hypoxanthine/aminopterin/thymidine medium. The majority (2/3) of the control hybrids grew in galactose as expected since Gal⁻ is dominant over Gal⁻. Fusion of Rhodamine 6-G treated Gal⁺TG^STK⁻ cells with Gal⁻32TG^RTK⁺ cells resulted in Rhodamine 6-G-tetraploid hybrids that were selected in hypoxanthine/aminopterin/thymidine medium. The majority (7/12) of the Rhodamine 6-G-hybrids grew in galactose as expected for a nuclearly encoded gene considering that Rhodamine 6-G interferes with transmission of mtDNA but not nuclear DNA. Therefore, these results are compelling in their demonstration of the nuclear origin of the Gal⁻ 32 mutation.     

Immunoglobulin lambda gene rearrangement can precede kappa gene rearrangement

Immunoglobulin genes are generated during differentiation of B lymphocytes by joining gene segments. A mouse pre-B cell contains a functional immunoglobulin heavy-chain gene, but no light-chain gene. Although there is only one heavy-chain locus, there are two light-chain loci: kappa and lambda. It has been reported that kappa loci in the germ-line configuration are never (in man) or very rarely (in the mouse) present in cells with functionally rearranged lambda-chain genes. Two explanations have been proposed to explain this: (a) the ordered rearrangement theory, which postulates that light-chain gene rearrangement in the pre-B cell is first attempted at the kappa locus, and that only upon failure to produce a functional kappa chain is there an attempt to rearrange the lambda locus; and (b) the stochastic theory, which postulates that rearrangement at the lambda locus proceeds at a rate that is intrinsically much slower than that at the kappa locus. We show here that lambda-chain genes are generated whether or not the kappa locus has lost its germ-line arrangement, a result that is compatible only with the stochastic theory.     

Dual gene targeted multimeric siRNA for combinatorial gene silencing

Simultaneous silencing of multiple up-regulated genes is an attractive and viable strategy to treat many incurable diseases including cancer. Herein we report that multimerized siRNA conjugate composed of two different siRNA sequences in the same backbone shows more efficient inhibition of the two corresponding target genes at one time than physically mixed multimerized siRNA conjugates. Two model siRNAs against VEGF and GFP gene were chemically crosslinked via cleavable and noncleavable linkages for the preparation of dual gene targeted multimeric siRNA conjugates (DGT multi-siRNA). Cleavable DGT multi-siRNA with reducible disulfide linkages exhibited significantly higher gene silencing efficiencies at mRNA and protein expression levels than noncleavable DGT multi-siRNA, the physical mixture of naked siRNA, and that of single gene targeted multimeric siRNA (SGT multi-siRNA) with eliciting negligible immune response. DGT multi-siRNAs against two therapeutic siRNAs, anti-survivin and anti-bcl-2 targeted siRNA, also showed greatly enhanced apoptotic effect. This approach for concurrent suppression of combinatorial therapeutic target genes using cleavable multimeric siRNA structure can be potentially used for improved therapeutic efficacy.