利用RNA-seq数据分析肝细胞癌差异表达基因及蛋白相互作用

RNA-Seq已成为当前转录组学研究的强有力工具,尤其在肿瘤差异表达基因的筛选方面有重要的应用价值。为进一步阐明肝细胞癌(HCC)的分子机制,本研究对GEO中1个包括12对HCC组织标本的RNA-Seq数据集(GSE63863)进行了生物信息学分析。采用edgeR、DESeq2、voom等3种不同算法的软件进行统计分析,共获得976个差异表达基因(adj. p-value<0.01或FDR<0.01,|logFC|≥2),其中上调表达422个(43.2%),下调554个(56.8%)。GO富集分析显示这些差异表达基因主要涉及离子结合、氧化还原酶活性等分子功能以及氧化还原、细胞分裂等生物学过程;KEGG通路分析显示,这些差异表达基因主要涉及细胞周期、视黄醇等代谢通路。STRING分析显示,共有654个基因编码的蛋白质存在相互作用,进一步利用MCODE分析显示,169个基因编码蛋白构成4个子网络,相应的中心节点基因分别为UBE2C、GNG4、TTR、FOS,这些基因的异常表达可能在HCC的发生发展过程中具有重要作用。上述研究结果将为进一步阐明HCC分子发病机制、寻找新型生物标志物提供初步的依据。     

肝细胞癌相关差异基因的生物信息学及预后分析

肝细胞癌是全球癌症相关死亡的主要原因,目前对肝细胞癌的发病机制研究尚不完善,探索肝细胞癌发生、发展相关的分子标志物及其预后具有重要意义。从GEO数据库获得肝细胞癌组织和非癌组织的基因表达阵列数据GSE84402,利用GEO2R筛选差异表达基因;采用DAVID数据库对差异基因进行GO富集分析和KEGG通路分析;通过STRING数据库和Cytoscape软件构建差异表达基因对应的蛋白质相互作用网络,并从网络中筛选出核心基因(hub genes);结合KM plotter数据库的临床信息对hub genes进行预后分析。结果显示:共得到1 307个差异表达基因,其中上调基因741个,下调基因566个,这些差异表达基因主要涉及细胞分裂、细胞周期、DNA复制及物质代谢等生物学过程及生物通路。通过GO、KEGG及蛋白质相互作用网络筛选出BUB1、BUB1B、CCNA2、CCNB1、CCNB2、CDC20、CDK1、MAD2L1、PLK1等9个hub genes,进一步分析发现hub genes均与细胞周期的调控相关,表明细胞周期的调控失常在肝细胞癌的发生、发展过程中具有重要作用。生存分析显示9个hub genes在肝细胞癌患者中均为表达上调的基因,且与患者预后不良相关,这为寻找肝细胞癌患者预后相关生物标志物的研究提供了线索。     

A Genome-wide Expression Association Analysis Identifies Genes and Pathways Associated with Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with strong genetic components. To identity novel risk variants for ALS, utilizing the latest genome-wide association studies (GWAS) and eQTL study data, we conducted a genome-wide expression association analysis by summary data-based Mendelian randomization (SMR) method. Summary data were derived from a large-scale GWAS of ALS, involving 12577 cases and 23475 controls. The eQTL annotation dataset included 923,021 cis-eQTL for 14,329 genes and 4732 trans-eQTL for 2612 genes. Genome-wide single gene expression association analysis was conducted by SMR software. To identify ALS-associated biological pathways, the SMR analysis results were further subjected to gene set enrichment analysis (GSEA). SMR single gene analysis identified one significant and four suggestive genes associated with ALS, including C9ORF72 (P value = 7.08 × 10^?6), NT5C3L (P value = 1.33 × 10^?5), GGNBP2 (P value = 1.81 × 10^?5), ZNHIT3(P value = 2.94 × 10^?5), and KIAA1600(P value = 9.97 × 10^?5). GSEA identified 7 significant biological pathways, such as PEROXISOME (empirical P value = 0.006), GLYCOLYSIS_GLUCONEOGENESIS (empirical P value = 0.043), and ARACHIDONIC_ACID_ METABOLISM (empirical P value = 0.040). Our study provides novel clues for the genetic mechanism studies of ALS.     

肝细胞癌高表达基因cDNA文库的构建及生物信息学分析

目的:构建肝细胞癌的高表达基因cDNA文库并进行相应的生物信息学分析。方法:应用抑制性消减杂交(SSH)技术,以7对肝癌组织和癌旁组织为实验材料,构建肝癌高表达基因的cDNA文库;使用BioEdit、BLAST和EGAD等软件进行生物信息学分析。结果:获得1450个白色阳性克隆,经PCR扩增后均有100～1000bb的插入片段,经杂交验证选取125个克隆进行测序;经BLAST、EGAD等生物信息学工具分析获得基因83个,其中细胞分裂相关基因5个,参与细胞信号转导或通讯的基因5个,参与细胞结构或运动的基因7个,细胞或器官防御相关基因11个,参与细胞内基因转录或蛋白表达的基因22个,代谢相关基因18个,另有15个未归类基因。结论:利用SSH技术可构建肝细胞癌组织差异表达基因的消减cDNA文库,为进一步深入探讨肝癌的诊断、治疗和预后评估等提供更多的分子指标。     

Integrated Enrichment Analysis of Variants and Pathways in Genome-Wide Association Studies Indicates Central Role for IL-2 Signaling Genes in Type 1 Diabetes,and Cytokine Signaling Genes in Crohn's Disease

Pathway analyses of genome-wide association studies aggregate information over sets of related genes, such as genes in common pathways, to identify gene sets that are enriched for variants associated with disease. We develop a model-based approach to pathway analysis, and apply this approach to data from the Wellcome Trust Case Control Consortium (WTCCC) studies. Our method offers several benefits over existing approaches. First, our method not only interrogates pathways for enrichment of disease associations, but also estimates the level of enrichment, which yields a coherent way to promote variants in enriched pathways, enhancing discovery of genes underlying disease. Second, our approach allows for multiple enriched pathways, a feature that leads to novel findings in two diseases where the major histocompatibility complex (MHC) is a major determinant of disease susceptibility. Third, by modeling disease as the combined effect of multiple markers, our method automatically accounts for linkage disequilibrium among variants. Interrogation of pathways from eight pathway databases yields strong support for enriched pathways, indicating links between Crohn''s disease (CD) and cytokine-driven networks that modulate immune responses; between rheumatoid arthritis (RA) and “Measles” pathway genes involved in immune responses triggered by measles infection; and between type 1 diabetes (T1D) and IL2-mediated signaling genes. Prioritizing variants in these enriched pathways yields many additional putative disease associations compared to analyses without enrichment. For CD and RA, 7 of 8 additional non-MHC associations are corroborated by other studies, providing validation for our approach. For T1D, prioritization of IL-2 signaling genes yields strong evidence for 7 additional non-MHC candidate disease loci, as well as suggestive evidence for several more. Of the 7 strongest associations, 4 are validated by other studies, and 3 (near IL-2 signaling genes RAF1, MAPK14, and FYN) constitute novel putative T1D loci for further study.     

基于生物信息学分析的肝癌核心基因的筛选及验证

本研究基于GEO数据库,选取由慢性乙型肝炎诱导的肝细胞癌芯片数据GSE121248为研究对象,利用GEO2R软件分析数据,筛选出差异表达基因,利用DAVID数据库进行GO分析和KEGG pathway富集分析.利用STRING数据库构建PPI网络,分析筛选核心基因.利用GEPIA对核心基因的表达进行验证,Kaplan ...     

Multi-SNP Analysis of GWAS Data Identifies Pathways Associated with Nonalcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is a common liver disease; the histological spectrum of which ranges from steatosis to steatohepatitis. Nonalcoholic steatohepatitis (NASH) often leads to cirrhosis and development of hepatocellular carcinoma. To better understand pathogenesis of NAFLD, we performed the pathway of distinction analysis (PoDA) on a genome-wide association study dataset of 250 non-Hispanic white female adult patients with NAFLD, who were enrolled in the NASH Clinical Research Network (CRN) Database Study, to investigate whether biologic process variation measured through genomic variation of genes within these pathways was related to the development of steatohepatitis or cirrhosis. Pathways such as Recycling of eIF2:GDP, biosynthesis of steroids, Terpenoid biosynthesis and Cholesterol biosynthesis were found to be significantly associated with NASH. SNP variants in Terpenoid synthesis, Cholesterol biosynthesis and biosynthesis of steroids were associated with lobular inflammation and cytologic ballooning while those in Terpenoid synthesis were also associated with fibrosis and cirrhosis. These were also related to the NAFLD activity score (NAS) which is derived from the histological severity of steatosis, inflammation and ballooning degeneration. Eukaryotic protein translation and recycling of eIF2:GDP related SNP variants were associated with ballooning, steatohepatitis and cirrhosis. Il2 signaling events mediated by PI3K, Mitotic metaphase/anaphase transition, and Prostanoid ligand receptors were also significantly associated with cirrhosis. Taken together, the results provide evidence for additional ways, beyond the effects of single SNPs, by which genetic factors might contribute to the susceptibility to develop a particular phenotype of NAFLD and then progress to cirrhosis. Further studies are warranted to explain potential important genetic roles of these biological processes in NAFLD.     

Quantitative Phosphoproteomic Analysis Identifies Activation of the RET and IGF-1R/IR Signaling Pathways in Neuroblastoma

Neuroblastoma is an embryonal tumor of childhood with a heterogenous clinical presentation that reflects differences in activation of complex biological signaling pathways. Protein phosphorylation is a key component of cellular signal transduction and plays a critical role in processes that control cancer cell growth and survival. We used shotgun LC/MS to compare phosphorylation between a human MYCN amplified neuroblastoma cell line (NB10), modeling a resistant tumor, and a human neural precursor cell line (NPC), modeling a normal baseline neural crest cell. 2181 unique phosphorylation sites representing 1171 proteins and 2598 phosphopeptides were found. Protein kinases accounted for 6% of the proteome, with a predominance of tyrosine kinases, supporting their prominent role in oncogenic signaling pathways. Highly abundant receptor tyrosine kinase (RTK) phosphopeptides in the NB10 cell line relative to the NPC cell line included RET, insulin-like growth factor 1 receptor/insulin receptor (IGF-1R/IR), and fibroblast growth factor receptor 1 (FGFR1). Multiple phosphorylated peptides from downstream mediators of the PI3K/AKT/mTOR and RAS pathways were also highly abundant in NB10 relative to NPC. Our analysis highlights the importance of RET, IGF-1R/IR and FGFR1 as RTKs in neuroblastoma and suggests a methodology that can be used to identify potential novel biological therapeutic targets. Furthermore, application of this previously unexploited technology in the clinic opens the possibility of providing a new wide-scale molecular signature to assess disease progression and prognosis.     

Xanthohumol Inhibits Notch Signaling and Induces Apoptosis in Hepatocellular Carcinoma

Despite improvement in therapeutic strategies, median survival in advanced hepatocellular carcinoma (HCC) remains less than one year. Therefore, molecularly targeted compounds with less toxic profiles are needed. Xanthohumol (XN), a prenylated chalcone has been shown to have anti-proliferative effects in various cancers types in vitro. XN treatment in healthy mice and humans yielded favorable pharmacokinetics and bioavailability. Therefore, we determined to study the effects of XN and understand the mechanism of its action in HCC. The effects of XN on a panel of HCC cell lines were assessed for cell viability, colony forming ability, and cellular proliferation. Cell lysates were analyzed for pro-apoptotic (c-PARP and cleaved caspase-3) and anti-apoptotic markers (survivin, cyclin D1, and Mcl-1). XN concentrations of 5μM and above significantly reduced the cell viability, colony forming ability and also confluency of all four HCC cell lines studied. Furthermore, growth suppression due to apoptosis was evidenced by increased expression of pro-apoptotic and reduced expression of anti-apoptotic proteins. Importantly, XN treatment inhibited the Notch signaling pathway as evidenced by the decrease in the expression of Notch1 and HES-1 proteins. Ectopic expression of Notch1 in HCC cells reverses the anti-proliferative effect of XN as evidenced by reduced growth suppression compared to control. Taken together these results suggested that XN mediated growth suppression is appeared to be mediated by the inhibition of the Notch signaling pathway. Therefore, our findings warrants further studies on XN as a potential agent for the treatment for HCC.     

Polymorphisms in Genes of Tricarboxylic Acid Cycle Key Enzymes Are Associated with Early Recurrence of Hepatocellular Carcinoma

Alterations of activity and expression in tricarboxylic acid (TCA) cycle key enzymes have been indicated in several malignancies, including hepatocellular carcinoma (HCC). They play an important role in the progression of cancer. However, the impact of single nucleotide polymorphisms (SNPs) in genes encoding these key enzymes on the recurrence of HCC has not been investigated. In this study, we genotyped 17 SNPs in genes encoding TCA cycle key enzymes and analyzed their association with recurrence-free survival (RFS) in a cohort of 492 Chinese HCC patients by Cox proportional hazard model and survival tree analysis. We identified 7 SNPs in SDHC, SDHD, FH, and IDH2 genes to be significantly associated with the RFS of HCC patients. Moreover, all these SNPs were associated with the early recurrence (within 2 years after surgery) risk of diseases. Cumulative effect analysis showed that these SNPs exhibited a dose-dependent effect on the overall and early recurrence. Further stratified analysis suggested that number of risk genotypes modified the protective effect on HCC recurrence conferred by transcatheter arterial chemoembolization treatment. Finally, the survival tree analysis revealed that SNP rs10789859 in SDHD gene was the primary factor contributing to HCC recurrence in our population. To the best of our knowledge, we for the first time observed the association between SNPs in genes encoding TCA cycle key enzymes and HCC recurrence risk. Further observational and functional studies are needed to validate our findings and generalize its clinical usage.     

VarWalker: Personalized Mutation Network Analysis of Putative Cancer Genes from Next-Generation Sequencing Data

A major challenge in interpreting the large volume of mutation data identified by next-generation sequencing (NGS) is to distinguish driver mutations from neutral passenger mutations to facilitate the identification of targetable genes and new drugs. Current approaches are primarily based on mutation frequencies of single-genes, which lack the power to detect infrequently mutated driver genes and ignore functional interconnection and regulation among cancer genes. We propose a novel mutation network method, VarWalker, to prioritize driver genes in large scale cancer mutation data. VarWalker fits generalized additive models for each sample based on sample-specific mutation profiles and builds on the joint frequency of both mutation genes and their close interactors. These interactors are selected and optimized using the Random Walk with Restart algorithm in a protein-protein interaction network. We applied the method in >300 tumor genomes in two large-scale NGS benchmark datasets: 183 lung adenocarcinoma samples and 121 melanoma samples. In each cancer, we derived a consensus mutation subnetwork containing significantly enriched consensus cancer genes and cancer-related functional pathways. These cancer-specific mutation networks were then validated using independent datasets for each cancer. Importantly, VarWalker prioritizes well-known, infrequently mutated genes, which are shown to interact with highly recurrently mutated genes yet have been ignored by conventional single-gene-based approaches. Utilizing VarWalker, we demonstrated that network-assisted approaches can be effectively adapted to facilitate the detection of cancer driver genes in NGS data.     

抑制消减杂交法筛选原发性肝细胞癌中差异表达的基因及其生物学意义

为了筛选原发性肝细胞癌 (hepatocellularcarcinoma ,HCC)中差异表达的基因 ,以了解HCC发生发展的分子基础 ,选取了一例早期高分化肝癌标本作为材料 ,采用抑制消减杂交 (suppressionsubtractivehybridization ,SSH)技术 ,进行了前向及反向消减杂交 ,结合反向Northern印迹筛选 ,得到多个差异表达的基因 .对有意义的基因用半定量RT PCR检测了肝癌中的表达 .结果显示 ,PON2、hSRP1alpha、H4 1在大部分肝癌中表达升高 ,IGFBP1、ITIH1在早期癌症中 ,大部分癌的表达升高 ,在晚期癌症中则表达下降 .EGR1在大部分肝癌中表达降低 .研究表明 ,不同分化程度、不同临床分期的肝癌 ,有共同的或不同的基因表达发生改变 ,明确这些差异表达的基因谱 ,对于肝癌发生发展机理的阐明及肝癌的预防、诊断、治疗都有重要意义 .     

Exploration of Involved Key Genes and Signaling Diversity in Brain Tumors

Brain tumors are becoming a major cause of death. The classification of brain tumors has gone through restructuring with regard to some criteria such as the presence or absence of a specific genetic alteration in the 2016 central nervous system World Health Organization update. Two categories of genes with a leading role in tumorigenesis and cancer induction include tumor suppressor genes and oncogenes; tumor suppressor genes are inactivated through a variety of mechanisms that result in their loss of function. As for the oncogenes, overexpression and amplification are the most common mechanisms of alteration. Important cell cycle genes such as p53, ATM, cyclin D2, and Rb have shown altered expression patterns in different brain tumors such as meningioma and astrocytoma. Some genes in signaling pathways have a role in brain tumorigenesis. These pathways include hedgehog, EGFR, Notch, hippo, MAPK, PI3K/Akt, and WNT signaling. It has been shown that telomere length in some brain tumor samples is shortened compared to that in normal cells. As the shortening of telomere length triggers chromosome instability early in brain tumors, it could lead to initiation of cancer. On the other hand, telomerase activity was positive in some brain tumors. It is suggestive that telomere length and telomerase activity are important diagnostic markers in brain tumors. This review focuses on brain tumors with regard to the status of oncogenes, tumor suppressors, cell cycle genes, and genes in signaling pathways as well as the role of telomere length and telomerase in brain tumors.