A computational approach to measuring coherence of gene expression in pathways

This study uses a computational approach to analyze coherence of expression of genes in pathways. Microarray data were analyzed with respect to coherent gene expression in a group of genes defined as a pathway in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Our hypothesis is that genes in the same pathway are more likely to be coordinately regulated than a randomly selected gene set. A correlation coefficient for each pair of genes in a pathway was estimated based on gene expression in normal or tumor samples, and statistically significant correlation coefficients were identified. The coherence indicator was defined as the ratio of the number of gene pairs in the pathway whose correlation coefficients are significant, divided by the total number of gene pairs in the pathway. We defined all genes that appeared in the KEGG pathways as a reference gene set. Our analysis indicated that the mean coherence indicator of pathways is significantly larger than the mean coherence indicator of random gene sets drawn from the reference gene set. Thus, the result supports our hypothesis. The significance of each individual pathway of n genes was evaluated by comparing its coherence indicator with coherence indicators of 1000 random permutation sets of n genes chosen from the reference gene set. We analyzed three data sets: two Affymetrix microarrays and one cDNA microarray. For each of the three data sets, statistically significant pathways were identified among all KEGG pathways. Seven of 96 pathways had a significant coherence indicator in normal tissue and 14 of 96 pathways had a significant coherence indicator in tumor tissue in all three data sets. The increase in the number of pathways with significant coherence indicators may reflect the fact that tumor cells have a higher rate of metabolism than normal cells. Five pathways involved in oxidative phosphorylation, ATP synthesis, protein synthesis, or RNA synthesis were coherent in both normal and tumor tissue, demonstrating that these are essential genes, a high level of expression of which is required regardless of cell type.     

Musashi1: A stem cell marker no longer in search of a function

One of the earliest genes identified with stem and early progenitor cells is the RNA-binding protein, Musashi1 (Msi1). Through gene profiling of mammary epithelial cells transduced with Msi1, a unique autocrine signaling pathway was identified that activates both the Wnt and Notch pathways 1. This process was associated with increased secretion of the growth factor, PLF1 and inhibition of the secreted Wnt pathway inhibitor, DKK3. Identification of PLF1 as an effector of these pathways in the absence of the DKK3 tumor suppressor provides a new avenue for investigating differences between normal and malignant tissues, and potentially targeting tumor stem cells.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase promoter by nuclear receptors liver receptor homologue-1 and small heterodimer partner: a mechanism for differential regulation of cholesterol synthesis and uptake

Cholesterol homeostasis in mammals involves pathways for biosynthesis, cellular uptake, and hepatic conversion to bile acids. Key genes for all three pathways are regulated by negative feedback control. Uptake and biosynthesis are directly regulated by cholesterol through its inhibition of the proteolytic activation of the sterol regulatory element binding proteins. The conversion of cholesterol into bile acids in the liver is regulated through the bile acid-dependent induction of the negatively acting small heterodimer partner nuclear receptor. In this report, we have shown that the small heterodimer partner also directly regulates cholesterol biosynthesis through inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase but has no effect on low density lipoprotein receptor expression. This has significant metabolic significance, as it provides both a mechanism to independently regulate cholesterol synthesis from uptake (an essential regulatory feature known to occur in vivo) and a pathway for direct regulation of cholesterol biosynthesis by bile acids. This latter feature ensures that the early phase of bile acid synthesis (pre-cholesterol) is in metabolic communication with the later stages of the pathway to properly regulate whole pathway flux. This highlights an important regulatory feature that is shared with other key branched, multienzyme pathways, such as glycolysis, where pathway outflow through pyruvate kinase is regulated by the concentration of a key early intermediate, fructose 1,6-bisphosphate.     

基于蛋白质互作知识的生物学通路扩充新方法

生物学通路被广泛应用于基因功能学研究, 但现有的生物学通路知识并不完善, 仍需进一步扩充。生物信息学预测为通路扩充提供了一种有效且经济的途径。文章提出了一种融合蛋白质-蛋白质互作知识以及Gene Ontology(GO)数据库信息进行基因通路预测的新方法。首先选取目标基因在蛋白质-蛋白质互作层面上的邻居所在的Kyoto Encyclopedia of Genes and Genomes(KEGG)通路为候选通路, 然后通过检验候选通路中的基因是否在与目标基因关联的GO节点富集来判断目标基因的通路归属。分别利用Human Protein Reference Database (HPRD)和Biological General Repository for Interaction Datasets(BioGRID)数据库中的蛋白质-蛋白质互作信息进行预测。结果表明, 在两套数据中, 随着互作邻居个数的增加, 预测的平均准确率(在所有目标基因注释的通路中被成功预测的比例)及相对准确率(在至少有一个注释通路被成功预测的基因集中, 所有注释通路均被预测正确的基因所占的比例)均呈现上升趋势。当互作邻居个数达到22时, 预测的平均准确率分别达到96.2%(HPRD)和96.3%(BioGRID), 而相对准确率分别为93.3%(HPRD)和84.1%(BioGRID)。进一步利用新版数据库对旧版数据库中被更新的89个基因进行验证, 至少有一个更新通路被预测正确的基因有50个, 其中43个基因的更新通路被完全正确预测, 相对准确率为86.0%。这些结果显示该方法是一种可靠且有效的通路扩充方法。     

Up-regulation of the Wnt, estrogen receptor, insulin-like growth factor-I, and bone morphogenetic protein pathways in C57BL/6J osteoblasts as opposed to C3H/HeJ osteoblasts in part contributes to the differential anabolic response to fluid shear

C57BL/6J (B6), but not C3H/HeJ (C3H), mice responded to mechanical loading with an increase in bone formation. A 30-min steady fluid shear of 20 dynes/cm(2) increased [(3)H]thymidine incorporation and alkaline phosphatase activity and up-regulated the expression of early mechanoresponsive genes (integrin beta1 (Igtb1) and cyclooxygenase-2 (Cox-2)) in B6 but not C3H osteoblasts, indicating that the differential mechanosensitivity was intrinsic to osteoblasts. In-house microarray analysis with 5,500 gene fragments revealed that the expression of 669 genes in B6 osteoblasts and 474 genes in C3H osteoblasts was altered 4 h after the fluid shear. Several genes associated with the insulin-like growth factor (IGF)-I, the estrogen receptor (ER), the bone morphogenetic protein (BMP)/transforming growth factor-beta, and Wnt pathways were differentially up-regulated in B6 osteoblasts. In vitro mechanical loading also led to up-regulation of these genes in the bones of B6 but not C3H mice. Pretreatment of B6 osteoblasts with inhibitors of the Wnt pathway (endostatin), the BMP pathway (Noggin), or the ER pathway (ICI182780) blocked the fluid shear-induced proliferation. Inhibition of integrin and Cox-2 activation by echistatin and indomethacin, respectively, each blocked the fluid shear-induced up-regulation of genes associated with these four pathways. In summary, up-regulation of the IGF-I, ER, BMP, and Wnt pathways is involved in mechanotransduction. These four pathways are downstream to the early mechanoresponsive genes, i.e. Igtb1 and Cox-2. In conclusion, differential up-regulation of these anabolic pathways may in part contribute to the good and poor response, respectively, in the B6 and C3H mice to mechanical loading.     

一种基于基因拓扑重要性的通路识别方法

癌症相关通路的识别是认识癌症发生发展过程机制的生物学基础。已有的通路识别方法很少考虑基因在通路中的拓扑重要性。重叠基因降权(PADOG)方法在基因集分析(GSA)方法的基础上融入了基因特异性的影响,提高了癌症相关通路的识别性能。为进一步提高癌症相关通路的识别性能,首先统计了KEGG通路数据集中基因出度的分布情况,根据基因出度的大小定义了基因的重要性。最后将基因的特异性和重要性融合在一起,提出了一种基于基因重要性和特异性的通路分析方法 PAGIS。在结肠癌、肺癌和胰腺癌3个数据集上的实验结果表明,PAGIS方法比PADOG能够提高很多癌症相关的排名,从而提高癌症相关通路的识别效果。     

茶足柄瘤蚜茧蜂蛹滞育过程中胰岛素信号通路及其相关途径的初探

本实验通过探索胰岛素信号通路及其相关途径对茶足柄瘤蚜茧蜂蛹滞育的影响,从而方便寻找胰岛素替代物,为害虫防治提供新思路。利用RNA-Seq,对滞育组与非滞育组的茶足柄瘤蚜茧蜂进行转录组测序,结合生物信息学方法对转录组中胰岛素信号通路及其相关途径的差异表达基因进行了分析。与胰岛素信号通路相关差异表达基因共31个,重点分析的PI3K-Akt, FoxO, MAPK三条途径,差异表达基因分别为55, 21和28个。这些滞育关联基因呈现不同程度的上调或下调表达,发现Sos, FASN, TSC1, PRKAB等基因与茶足柄瘤蚜茧蜂滞育密切相关,共同影响茶足柄瘤蚜茧蜂的滞育。胰岛素信号通路及其相关途径对茶足柄瘤蚜茧蜂的滞育起着非常重要的作用,主要体现在影响虫体能量代谢、脂质积累、细胞增殖等方面。     

基于蛋白互作网络识别非小细胞肺癌相关基因功能模块

本研究对非小细胞肺癌(non-small cell lung carcinoma,NSCLC)基因表达数据进行差异表达分析,并与蛋白质相互作用网络(PPIN)数据进行整合,进一步利用Heinz搜索算法识别NSCLC相关的基因功能模块,并对模块中的基因进行功能(GO term)和通路(KEGG)富集分析,旨在探究肺癌发病分子机制。蛋白互作网络分析得到一个包含96个基因和117个相互作用的功能模块,以及8个对NSCLC的发生和发展起到关键作用候选基因标志物。富集分析结果表明,这些基因主要富集于基因转录催化及染色质调控等生物学过程,并在基础转录因子、黏着连接、细胞周期、Wnt信号通路及HTLV-Ⅰ感染等生物学通路中发挥重要作用。本研究对非小细胞肺癌相关的基因和生物学通路进行预测,可用于肺癌的早期诊断和早期治疗,以降低肺癌死亡率。     

A protein kinase A and Wnt-dependent network regulating an intermediate stage in epithelial tubulogenesis during kidney development

Genetic interactions regulating intermediate stages of tubulogenesis in the developing kidney have been difficult to define. A systems biology strategy using microarray was combined with in vitro/ex vivo and genetic approaches to identify pathways regulating specific stages of tubulogenesis. Analysis of the progression of the metanephric mesenchyme (MM) through four stages of tubule induction and differentiation (i.e., epithelialization, tubular organization and elongation and early differentiation) revealed signaling pathways potentially involved at each stage and suggested key roles for a number of signaling molecules. A screen of the signaling pathways on in vitro/ex vivo nephron formation implicated a unique regulatory role for protein kinase A (PKA), through PKA-2, in a specific post-epithelialization morphogenetic step (conversion of the renal vesicle to the S-shaped body). Microarray analysis not only confirmed this stage-specificity, but also highlighted the upregulation of Wnt genes. Addition of PKA agonists to LIF-induced nephrons (previously shown to be a Wnt/beta-catenin dependent pathway) disrupted normal tubulogenesis in a manner similar to PKA-agonist treated MM/spinal-cord assays, suggesting that PKA regulates a Wnt-dependent tubulogenesis step. PKA induction of canonical Wnt signaling during tubulogenesis was confirmed genetically using MM from Batgal-reporter mice. Addition of a Wnt synthesis inhibitor to activated PKA cultures rescued tubulogenesis. By re-analysis of existing microarray data from the FGF8, Lim1 and Wnt4 knockouts, which arrest in early tubulogenesis, a network of genes involving PKA, Wnt, Lhx1, FGF8, and hyaluronic acid signaling regulating the transition of nascent epithelial cells to tubular epithelium was derived, helping to reconcile in vivo and in vitro/ex vivo data.     

A putative twin-arginine translocation pathway in Legionella pneumophila

Legionella pneumophila is a facultative intracellular human pathogen causing Legionnaires' disease, a severe form of pneumonia. Because of the importance of secretion pathways in virulence, we were interested in the possible presence of the twin-arginine translocation (Tat) pathway in L. pneumophila. This secretion pathway is used to transport folded proteins, characterized by two arginines in their signal peptide, across the cytoplasmic membrane. We describe here the presence of a putative Tat pathway in L. pneumophila. Three genes encoding Escherichia coli TatA, TatB, and TatC homologues were identified. The tatA and tatB genes were shown to constitute an operon while tatC is monocistronic. RT-PCR analysis revealed expression of the tat genes during both exponential and stationary growth as well as during intracellular growth in Acanthamoeba castellanii. A search for the conserved twin-arginine motif in predicted signal peptides resulted in a list of putative Tat substrates.     

Gene expression analysis of toxicological pathways in TM3 leydig cell lines treated with Ethane dimethanesulfonate

Ethane dimethanesulfonate (EDS), a well-known alkylating agent, selectively destroys Leydig cells. To clarify the molecular pathways underlying EDS action on Leydig cells, we analyzed gene expression profiles of an EDS-treated TM3 Leydig cell line. In this study, we analyzed the representative canonical pathways and toxicity pathways/gene lists using the Ingenuity Pathways Analysis program. In TM3 cells, 677 and 6756 genes were identified as being up- or downregulated after 3 and 24 h EDS treatments, respectively, (>1.3-fold changes, p < 0.05). Toxicological pathway analysis revealed that expression of genes related to Nrf2-mediated oxidative stress response showed remarkable changes in early or later stage of EDS-treated TM3 cells. Several genes related to steroidogenesis and apoptosis were also differentially expressed at 24 h in EDS-treated TM3 cells. Overall, toxicological pathway analysis using gene expression profiling showed that oxidative stress might be an important factor in cell death in TM3 cells affected by EDS treatment.     

Colony-stimulating factor-1 receptor utilizes multiple signaling pathways to induce cyclin D2 expression

Colony-stimulating factor-1 (CSF-1) induces expression of immediate early gene, such as c-myc and c-fos and delayed early genes such as D-type cyclins (D1 and D2), whose products play essential roles in the G1 to S phase transition of the cell cycle. Little is known, however, about the cytoplasmic signal transduction pathways that connect the surface CSF-1 receptor to these genes in the nucleus. We have investigated the signaling mechanism of CSF-1-induced D2 expression. Analyses of CSF-1 receptor autophosphorylation mutants show that, although certain individual mutation has a partial inhibitory effect, only multiple combined mutations completely block induction of D2 in response to CSF-1. We report that at least three parallel pathways, the Src pathway, the MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, and the c-myc pathway, are involved. Induction of D2 is partially inhibited in Src(-/-) bone marrow-derived macrophages and by Src inhibitor PP1 and is enhanced in v-Src-overexpressing cells. Activation of myc's transactivating activity selectively induces D2 but not D1. Blockade of c-myc expression partially blocks CSF-1-induced D2 expression. Complete inhibition of the MEK/ERK pathway causes 50% decrease of D2 expression. Finally, simultaneous inhibition of Src, MEK activation, and c-myc expression additively blocks CSF-1-induced D2 expression. This study indicates that multiple signaling pathways are involved in full induction of a single gene, and this finding may also apply broadly to other growth factor-inducible genes.     

Establishment of mouse teratocarcinomas stem cells line and screening genes responsible for malignancy

The sequential transplantation of embryonal carcinoma cells in vivo can accelerate the growth and malignancy of teratocarcinomas. However, the possible molecular mechanisms in this process that reflect cancer formation in the early stage are largely unknown and. To identify which genes are associated with the changes of malignancy of teratocarcinomas, we established a tumorigenesis model in which teratocarcinoma were induced via injecting embryonic stem cells into immuno-deficiency mice, isolating teratocarcinoma stem cell from a teratocarcinoma in serum-free culture medium and injecting teratocarcinoma stem cells into immune-deficient mice continuously. By using high-throughput deep sequence technology, we identified 26 differentially expressed genes related to the changes of characteristics of teratocarcinoma stem cell in which 18 out of 26 genes were down-regulated and 8 genes were up-regulated. Among these genes, several tumor-related genes such as Gata3, Arnt and Tdgf1, epigenetic associated genes such as PHC1 and Uty were identified. Pathway enrichment analysis result revealed that Wnt signaling pathway, primary immunodeficiency pathway, antigen processing and presentation pathway and allograft rejection pathway were involved in the teratocarcinoma tumorigenesis (corrected p value<0.05). In summary, our study established a tumorigenesis model and proposed some candidate genes and signaling pathways that may play a key role in the early stage of cancer occurrence.     

Multiple and interconnected pathways for L-lysine catabolism in Pseudomonas putida KT2440

L-lysine catabolism in Pseudomonas putida KT2440 was generally thought to occur via the aminovalerate pathway. In this study we demonstrate the operation of the alternative aminoadipate pathway with the intermediates D-lysine, L-pipecolate, and aminoadipate. The simultaneous operation of both pathways for the use of L-lysine as the sole carbon and nitrogen source was confirmed genetically. Mutants with mutations in either pathway failed to use L-lysine as the sole carbon and nitrogen source, although they still used L-lysine as the nitrogen source, albeit at reduced growth rates. New genes were identified in both pathways, including the davB and davA genes that encode the enzymes involved in the oxidation of L-lysine to delta-aminovaleramide and the hydrolysis of the latter to delta-aminovalerate, respectively. The amaA, dkpA, and amaB genes, in contrast, encode proteins involved in the transformation of Delta1-piperidine-2-carboxylate into aminoadipate. Based on L-[U-13C, U-15N]lysine experiments, we quantified the relative use of pathways in the wild type and its isogenic mutants. The fate of 13C label of L-lysine indicates that in addition to the existing connection between the D- and L-lysine pathways at the early steps of the catabolism of L-lysine mediated by a lysine racemase, there is yet another interconnection at the lower end of the pathways in which aminoadipate is channeled to yield glutarate. This study establishes an unequivocal relationship between gene and pathway enzymes in the metabolism of L-lysine, which is of crucial importance for the successful colonization of the rhizosphere of plants by this microorganism.