A Functional Variant in the MTOR Promoter Modulates Its Expression and Is Associated with Renal Cell Cancer Risk

Background

The mTOR signaling pathway plays a crucial role in the carcinogenesis of renal cell cancer (RCC). We sought to investigate the influence of genetic variations in the mTOR pathway-related genes on the risk of RCC.

Methods

We genotyped 8 potentially functional polymorphisms in AKT1, AKT2, PTEN and MTOR genes using the TaqMan method in a case-control study of 710 RCC patients and 760 cancer-free subjects. Unconditional logistic regression, adjusted for potential confounding factors, was used to assess the risk associations. We then examined the functionality of the important polymorphisms.

Results

Of the 8 polymorphisms, after adjusting for multiple comparisons, we found a significant association between one variant (rs2295080) in the promoter of MTOR and reduced RCC risk (P = 0.005, OR = 0.74, 95%CI = 0.59–0.91, TG/GG vs. TT). Another variant (rs701848) in the 3′UTR region of PTEN was associated with increased RCC risk (P = 0.014, OR = 1.45, 95%CI = 1.08–1.96, CC vs. TT); however, the association was not significant after adjusting for multiple comparisons. Furthermore, we observed lower MTOR mRNA levels in the presence of the rs2295080G allele in normal renal tissues. The luciferase reporter assay showed that the rs2295080G allele significantly decreased luciferase activity. No other significant association between the selected polymorphisms and RCC risk was observed.

Conclusions

Our results suggest that the functional MTOR promoter rs2295080 variant affects RCC susceptibility by modulating the endogenous MTOR expression level. The risk effects and the functional impact of the MTOR rs2295080 variant need further validation.     

Evaluation of Schistosome Promoter Expression for Transgenesis and Genetic Analysis

Schistosome worms of the genus Schistosoma are the causative agents of schistosomiasis, a devastating parasitic disease affecting more than 240 million people worldwide. Schistosomes have complex life cycles, and have been challenging to manipulate genetically due to the dearth of molecular tools. Although the use of gene overexpression, gene knockouts or knockdowns are straight-forward genetic tools applied in many model systems, gene misexpression and genetic manipulation of schistosome genes in vivo has been exceptionally challenging, and plasmid based transfection inducing gene expression is limited. We recently reported the use of polyethyleneimine (PEI) as a simple and effective method for schistosome transfection and gene expression. Here, we use PEI-mediated schistosome plasmid transgenesis to define and compare gene expression profiles from endogenous and nonendogenous promoters in the schistosomula stage of schistosomes that are potentially useful to misexpress (underexpress or overexpress) gene product levels. In addition, we overexpress schistosome genes in vivo using a strong promoter and show plasmid-based misregulation of genes in schistosomes, producing a clear and distinct phenotype- death. These data focus on the schistosomula stage, but they foreshadow strong potential for genetic characterization of schistosome molecular pathways, and potential for use in overexpression screens and drug resistance studies in schistosomes using plasmid-based gene expression.     

Tau Modulates VGluT1 Expression

Tau displacement from microtubules is the first step in the onset of tauopathies and is followed by toxic protein aggregation. However, other non-canonical functions of Tau might have a role in these pathologies. Here, we demonstrate that a small amount of Tau localizes in the nuclear compartment and accumulates in both the soluble and chromatin-bound fractions. We show that favoring Tau nuclear translocation and accumulation, by Tau overexpression or detachment from MTs, increases the expression of VGluT1, a disease-relevant gene directly involved in glutamatergic synaptic transmission. Remarkably, the P301L mutation, related to frontotemporal dementia FTDP-17, impairs this mechanism leading to a loss of function. Altogether, our results provide the demonstration of a direct physiological role of Tau on gene expression. Alterations of this mechanism may be at the basis of the onset of neurodegeneration.     

甘蓝型油菜BnGOLS1基因启动子的克隆、序列分析及瞬时表达

以甘蓝型油菜‘德油五号’基因组DNA为模板,通过反向PCR扩增得到肌醇半乳糖苷合成酶基因（BnGOLS1）启动子片段,长度为827bp。PLACE和PlantCARE启动子预测工具分析表明：序列中含有TATA-Box、CAAT-Box等基本转录元件,以及ABRE、DRE、HSE、w-Box等顺式作用元件。将克隆得到的BnGOLS1启动子取代pBI121中的CaMV35S启动子,构建BnGOLS1启动子控制报告基因的GUS表达载体pBI-GS-GUS,通过农杆菌介导的方法在油菜组织中进行瞬时表达。GUS染色结果表明BnGOLS1启动子可以驱动GUS基因在油菜组织中的表达。     

Dopamine D1 Receptor Gene Variation Modulates Opioid Dependence Risk by Affecting Transition to Addiction

Dopamine D1 receptor (DRD1) modulates opioid reinforcement, reward, and opioid-induced neuroadaptation. We propose that DRD1 polymorphism affects susceptibility to opioid dependence (OD), the efficiency of transition to OD, and opioid-induced pleasure response. We analyzed potential association between seven DRD1 polymorphisms with the following traits: duration of transition from the first use to dependence (DTFUD), subjective pleasure responses to opioid on first use and post-dependence use, and OD risk in 425 Chinese with OD and 514 healthy controls. DTFUD and level of pleasure responses were examined using a semi-structured interview. The DTFUD of opioid addicts ranged from 5 days to 11 years. Most addicts (64.0%) reported non-comfortable response upon first opioid use, while after dependence, most addicts (53.0%) felt strong opioid-induced pleasure. Survival analysis revealed a correlation of prolonged DTFUD with the minor allele-carrying genotypes of DRD1 rs4532 (hazard ratios (HR) = 0.694; p = 0.001) and rs686 (HR = 0.681, p = 0.0003). Binary logistic regression indicated that rs10063995 GT genotype (vs. GG+TT, OR = 0.261) could predict decreased pleasure response to first-time use and the minor alleles of rs686 (OR = 0.535) and rs4532 (OR = 0.537) could predict decreased post-dependence pleasure. Moreover, rs686 minor allele was associated with a decreased risk for rapid transition from initial use to dependence (DTFUD≤30 days; OR = 0.603) or post-dependence euphoria (OR = 0.603) relative to major allele. In conclusion, DRD1 rs686 minor allele decreases the OD risk by prolonging the transition to dependence and attenuating opioid-induced pleasure in Chinese.     

转录因子XBP1S基因表达谱差异分析

目的 应用基因表达谱芯片技术了解XBP1S在肝细胞中可能上调或下调的基因,了解其可能的调节功能线索.方法 构建pcDNA3.1（-）-XBP1S真核表达载体,转染HepG2细胞,同时以空载体pcDNA3.1（-）处理相同细胞系作为对照.48 h后制备细胞裂解液,提取mRNA,应用基因表达谱芯片技术对差异表达mRNA进行检测和分析.结果 构建的表达载体经过限制性内切酶分析和DNA序列测定,证实准确无误,提取高质量的总mRNA并进行逆转录成为cDNA,进行基因表达谱芯片技术分析.经过差异基因表达谱的筛选,发现HepG2细胞转染XBP1S以后,有38个基因表达水平显著上调,30个基因表达水平显著下调.结论 成功构建XBP1S的真核表达载体pcDNA3.1（-）-XBP1S,运用基因表达谱芯片技术成功筛选了XBP1S转染细胞后的差异表达基因,这些差异表达基因包括细胞周期、蛋白质的翻译合成及运输、能量代谢、体内免疫调节、细胞凋亡及细胞内的信号转导等方面起重要作用及肿瘤发生相关的基因,为进一步阐明XBP1S可能存在的调控机制及XBP1S蛋白可能的生物学功能提供理论依据.     

Inhibin Alpha-Subunit (INHA) Expression in Adrenocortical Cancer Is Linked to Genetic and Epigenetic INHA Promoter Variation

Adrenocortical carcinoma (ACC) is a rare, but highly malignant tumor of unknown origin. Inhibin α-subunit (Inha) knockout mice develop ACCs following gonadectomy. In man, INHA expression varies widely within ACC tissues and its circulating peptide inhibin pro-αC has been described as a novel tumor marker for ACC. We investigated whether genetic and epigenetic changes of the INHA gene in human ACC cause loss or variation of INHA expression. To this end, analyses of INHA sequence, promoter methylation and mRNA expression were performed in human adrenocortical tissues. Serum inhibin pro-αC levels were also measured in ACC patients. INHA genetic analysis in 37 unique ACCs revealed 10 novel, heterozygous rare variants. Of the 3 coding bases affected, one variant was synonymous and two were missense variants: S72F and S184F. The minor allele of rs11893842 at −124 bp was observed at a low frequency (24%) in ACC samples and was associated with decreased INHA mRNA levels: 4.7±1.9 arbitrary units for AA, compared to 26±11 for AG/GG genotypes (P = 0.034). The methylation of four proximal INHA promoter CpGs was aberrantly increased in five ACCs (47.7±3.9%), compared to normal adrenals (18.4±0.6%, P = 0.0052), whereas the other 14 ACCs studied showed diminished promoter methylation (9.8±1.1%, P = 0.020). CpG methylation was inversely correlated to INHA mRNA levels in ACCs (r = −0.701, p = 0.0036), but not associated with serum inhibin pro-αC levels. In conclusion, aberrant methylation and common genetic variation in the INHA promoter occur in human ACCs and are associated with decreased INHA expression.     

Genetically Dependent ERBB3 Expression Modulates Antigen Presenting Cell Function and Type 1 Diabetes Risk

Type 1 diabetes (T1D) is an autoimmune disease resulting from the complex interaction between multiple susceptibility genes, environmental factors and the immune system. Over 40 T1D susceptibility regions have been suggested by recent genome-wide association studies; however, the specific genes and their role in the disease remain elusive. The objective of this study is to identify the susceptibility gene(s) in the 12q13 region and investigate the functional link to the disease pathogenesis. A total of 19 SNPs in the 12q13 region were analyzed by the TaqMan assay for 1,434 T1D patients and 1,865 controls. Thirteen of the SNPs are associated with T1D (best p = 4×10⁻¹¹), thus providing confirmatory evidence for at least one susceptibility gene in this region. To identify candidate genes, expression of six genes in the region was analyzed by real-time RT-PCR for PBMCs from 192 T1D patients and 192 controls. SNP genotypes in the 12q13 region are the main factors that determine ERBB3 mRNA levels in PBMCs. The protective genotypes for T1D are associated with higher ERBB3 mRNA level (p<10⁻¹⁰). Furthermore, ERBB3 protein is expressed on the surface of CD11c⁺ cells (dendritic cells and monocytes) in peripheral blood after stimulation with LPS, polyI:C or CpG. Subjects with protective genotypes have significantly higher percentages of ERBB3⁺ monocytes and dendritic cells (p = 1.1×10⁻⁹); and the percentages of ERBB3⁺ cells positively correlate with the ability of APC to stimulate T cell proliferation (R² = 0.90, p<0.0001). Our results indicate that ERBB3 plays a critical role in determining APC function and potentially T1D pathogenesis.     

利用DNA池技术研究猪GH基因启动子序列的多态性

目的:分析猪GH基因启动子区序列的多态性,期望筛选出对猪生长性状有显著影响的SNP位点,为地方猪种的选育及选种提供一定的理论依据.方法:以大约克、可乐猪、香猪和黔北黑猪为试验对象,构建品种DNA池,采用PCR产物直接测序法对猪GH基因启动子区-856～+171片段共1 027bp进行单核苷酸多态性检测.结果:除香猪外,在其他3个猪种5'-端侧翼序列发现5个SNP位点:C22T、A- 26T、G- 219A、T- 385A和C-391T,并且在大约克GH基因启动子区-640处发现了一个12bp碱基序列(GGCAAAGTGTAG)的缺失.结论:DNA池结合PCR产物直接测序技术能够很好的筛选SNP位点,本研究采用该技术在猪GH基因启动子区- 856～+171片段检测到了5个SNP位点.     

巴西橡胶树 HbNAC1基因启动子的克隆及序列分析

NAC转录因子是植物特有的一类转录调控因子,在植物的生长发育、激素调节和境胁迫应答中具有重要的功能。为研究NAC转录因子在巴西橡胶树(Hevea brasiliensis)抗逆胁迫中的功能,本项目组根据巴西橡胶树NAC转录因子-HbNAC1基因序列,通过Genome Walking方法从巴西橡胶基因组DNA中获得了长度为1861bp的HbNAC1基因的5’调控区片段,序列分析表明该段序列含有一个典型的真核生物核心启动子区域,转录起始位点T位于起始密码子上游52bp处。该启动子序列除了含有TATA-box、CAAT-box等基本顺式作用元件外,还具有茉莉酸响应元件以及大量光顺式作用元件和逆境胁迫诱导相关的顺式调控元件,这表明HbNAC1基因在橡胶树逆境胁迫应答过程具有重要功能,其启动子可能是一个光诱导型和组织特异性启动子。     

巴西橡胶树HbNTL1基因启动子的克隆与序列分析

膜结合NAC转录因子(NTLs)是植物NAC转录因子家族中一类C端具有跨膜结构域(transmembrane motifs,TMs)的转录调控因子,在植物生长发育、激素调节和逆境胁迫应答中具有重要的功能。根据巴西橡胶树(Hevea brasiliensis)膜结合类NAC转录因子HbNTL1基因cDNA序列,利用基因组步移的方法从巴西橡胶树叶片基因组DNA中克隆获得了HbNTL1基因上游1 718 bp的调控片段。序列分析表明,该段序列含有一个典型的真核生物核心启动子区域,转录起始位点A位于起始密码子上游206 bp处。该启动子序列除了含有多个TATA-box、CAAT-box等基本顺式作用元件外,还存在赤霉素、茉莉酸和脱落酸等激素响应元件以及大量逆境胁迫诱导相关的顺式调控元件,如ABRE、DOFCOREZM、MYBCORE、W-box和MYCCONSENSUSATHSE等反应元件,表明HbNTL1转录因子可能是一个逆境胁迫相关NAC转录因子,在橡胶树抵御逆境胁迫的生理过程中具有重要功能。     

棉花nodulin-like基因及其启动子的结构特征和遗传表达

用同尾酶反向PCR技术(Ⅱ-PCR)和快速分离目的基因cDNA 5'未知序列方法(RICUP)首次从陆地棉品种Y18(Gossypium arboreum L.Y18)中分离到nodulin-like基因的全长cDNA、DNA和启动子序列.结果表明,该基因全长为2 353 bp,具有5个内含子和6个外显子.cDNA全长1480 bp,含有一个1125 bp的ORF结构,编码375个氨基酸,在GenBank nr数据库中没有与之同源的基因序列,在EST数据库中仅有一条733 bp的亚洲棉纤维EST序列(GenBank登录号:BF271235)与之有92%的同源性.Nodulin-like基因的启动子全长1 969 bp,启动子区域具有Initiator、TATA box、CAAT-like box和富含AT序列等启动子特征序列.Southern blot结果表明:该基因在棉花基因组中有一个拷贝.Northern blot结果表明:该基因在棉花的蕾、花、纤维和铃壳等生殖器官中呈优势表达.本研究有望为改良棉花生殖器官的农艺性状提供靶基因,并为解决目前转基因抗虫棉"前期抗虫性强、后期抗虫性弱"这一生产实际问题提供有效的表达调控元件.