Synergistic Effect of Metformin and Lansoprazole Against Gastric Cancer through Growth Inhibition.

Cancer has been linked to metabolic disorders and diverse gene mutations. Metformin, which is widely used to treat type 2 diabetes, inhibits the growth of cancer cells in animal models. Here we investigated the effects of metformin on human gastric cancer cell lines. We also investigated the synergistic anticancer effect of metformin and proton pump inhibitors. Lansoprazole, a proton pump inhibitor, is effective for treating gastroesophageal reflux disease. Our results revealed that metformin and lansoprazole can significantly inhibit cancer cell growth in a dose-dependent manner by suppressing cell cycle progression and inducing apoptosis. Low concentrations of metformin and lansoprazole have a synergistic effect on AGS cell growth inhibition. In summary, our findings suggest a new and safe treatment protocol for treating stomach cancers.

Hepatitis B virus virion secretion is a CRM1-spike-mediated late event.

BACKGROUND: Hepatitis B virus (HBV) is a major human pathogen worldwide. To date, there is no curative treatment for chronic hepatitis B. The mechanism of virion secretion remains to be investigated. Previously, we found that nuclear export of HBc particles can be facilitated via two CRM1-specific nuclear export signals (NES) at the spike tip. METHODS: In this study, we used site-directed mutagenesis at the CRM1 NES, as well as treatment with CRM1 inhibitors at a low concentration, or CRM1-specific shRNA knockdown, in HBV-producing cell culture, and measured the secretion of various HBV viral and subviral particles via a native agarose gel electrophoresis assay. Separated HBV particles were characterized by Western blot analysis, and their genomic DNA contents were measured by Southern blot analysis. Secreted extracellular particles were compared with intracellular HBc capsids for DNA synthesis and capsid formation. Virion secretion and the in vivo interactions among HBc capsids, CRM1 and microtubules, were examined by proximity ligation assay, immunofluorescence microscopy, and nocodazole treatment. RESULTS: We report here that the tip of spike of HBV core (HBc) particles (capsids) contains a complex sensor for secretion of both HBV virions and naked capsids. HBV virion secretion is closely associated with HBc nuclear export in a CRM1-dependent manner. At the conformationally flexible spike tips of HBc particles, NES motifs overlap extensively with motifs important for secretion of HBV virions and naked capsids. CONCLUSIONS: We provided experimental evidence that virions and naked capsids can egress via two distinct, yet overlapping, pathways. Unlike the secretion of naked capsids, HBV virion secretion is highly CRM1- and microtubule-dependent. CRM1 is well known for its involvement in nuclear transport in literature. To our knowledge, this is the first report that CRM1 is required for virion secretion. CRM1 inhibitors could be a promising therapeutic candidate for chronic HBV patients in clinical medicine.

Chemical and Biochemical Aspects of Molecular Hydrogen in Treating Kawasaki Disease and COVID-19.

Kawasaki disease (KD) is a systemic vasculitis and is the most commonly acquired heart disease among children in many countries, which was first reported 50 years ago in Japan. The 2019 coronavirus disease (COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)) has been a pandemic in most of the world since 2020, and since late 2019 in China. Kawasaki-like disease caused by COVID-19 shares some symptoms with KD, referred to as multisystem inflammatory syndrome in children, and has been reported in the United States, Italy, France, England, and other areas of Europe, with an almost 6-10 times or more increase compared with previous years of KD prevalence. Hydrogen gas is a stable and efficient antioxidant, which has a positive effect on oxidative damage, inflammation, cell apoptosis, and abnormal blood vessel inflammation. This review reports the chemical and biochemical aspects of hydrogen gas inhalation in treating KD and COVID-19.

Covariate-adjusted heatmaps for visualizing biological data via correlation decomposition.

Motivation: Heatmap is a popular visualization technique in biology and related fields. In this study, we extend heatmaps within the framework of matrix visualization (MV) by incorporating a covariate adjustment process through the estimation of conditional correlations. MV can explore the embedded information structure of high-dimensional large-scale datasets effectively without dimension reduction. The benefit of the proposed covariate-adjusted heatmap is in the exploration of conditional association structures among the subjects or variables that cannot be done with conventional MV. Results: For adjustment of a discrete covariate, the conditional correlation is estimated by the within and between analysis. This procedure decomposes a correlation matrix into the within- and between-component matrices. The contribution of the covariate effects can then be assessed through the relative structure of the between-component to the original correlation matrix while the within-component acts as a residual. When a covariate is of continuous nature, the conditional correlation is equivalent to the partial correlation under the assumption of a joint normal distribution. A test is then employed to identify the variable pairs which possess the most significant differences at varying levels of correlation before and after a covariate adjustment. In addition, a z-score significance map is constructed to visualize these results. A simulation and three biological datasets are employed to illustrate the power and versatility of our proposed method. Availability and implementation: GAP is available to readers and is free to non-commercial applications. The installation instructions, the user's manual, and the detailed tutorials can be found at http://gap.stat.sinica.edu.tw/Software/GAP. Supplementary information: Supplementary Data are available at Bioinformatics online.

A Comprehensive Analysis of Transcript-Supported De Novo Genes in Saccharomyces sensu stricto Yeasts.

Novel genes arising from random DNA sequences (de novo genes) have been suggested to be widespread in the genomes of different organisms. However, our knowledge about the origin and evolution of de novo genes is still limited. To systematically understand the general features of de novo genes, we established a robust pipeline to analyze >20,000 transcript-supported coding sequences (CDSs) from the budding yeast Saccharomyces cerevisiae. Our analysis pipeline combined phylogeny, synteny, and sequence alignment information to identify possible orthologs across 20 Saccharomycetaceae yeasts and discovered 4,340 S. cerevisiae-specific de novo genes and 8,871 S. sensu stricto-specific de novo genes. We further combine information on CDS positions and transcript structures to show that >65% of de novo genes arose from transcript isoforms of ancient genes, especially in the upstream and internal regions of ancient genes. Fourteen identified de novo genes with high transcript levels were chosen to verify their protein expressions. Ten of them, including eight transcript isoform-associated CDSs, showed translation signals and five proteins exhibited specific cytosolic localizations. Our results suggest that de novo genes frequently arise in the S. sensu stricto complex and have the potential to be quickly integrated into ancient cellular network.

Clinical significance of circulating plasma DNA in gastric cancer.

With the progression of molecular techniques, the detection of circulating plasma DNA (cpDNA) is clinically feasible. However, the role of the cpDNA levels in gastric cancer is not well understood. This study assessed the mutational profile in primary tumors and clarified the clinical utility of quantitative and qualitative cpDNA alterations in 277 patients with advanced gastric cancer. The concentrations of cpDNA were measured by TaqMan qPCR, and 68 mutations in 8 genes were studied for cpDNA mutations. The median cpDNA concentrations in patients with stages I, II, and III gastric cancer were 3979, 3390 and 4278 copies/mL, respectively, and increased to 11,380 copies/mL in patients with Stage IV gastric cancer (p < 0.001). Among the 35 patients harboring cpDNA mutations, Stage IV patients (100%) were more likely to display high cpDNA levels than were Stage I (33.3%), II (75%) and III patients (66.7%) (p = 0.037). Patients displaying high cpDNA levels were more likely to experience peritoneal recurrence and exhibited significantly lower 5-year overall survival rates (39.2% vs. 45.8%, p = 0.039) than did patients displaying low cpDNA levels. Only for late stage (Stages III or IV) gastric cancer, patients harboring cpDNA mutations were more likely to experience vascular invasion (20% vs. 2.4%, p = 0.036) and exhibited a lower 5-year overall survival rate than did those lacking cpDNA mutations (5.6% vs. 31.5%, p = 0.028). High cpDNA levels are associated with peritoneal recurrence and poor prognosis in patients with advanced gastric cancer; harboring cpDNA mutations is associated with poor prognosis among patients with late stage gastric cancer.

Smoking-related microRNAs and mRNAs in human peripheral blood mononuclear cells.

Teenager smoking is of great importance in public health. Functional roles of microRNAs have been documented in smoke-induced gene expression changes, but comprehensive mechanisms of microRNA-mRNA regulation and benefits remained poorly understood. We conducted the Teenager Smoking Reduction Trial (TSRT) to investigate the causal association between active smoking reduction and whole-genome microRNA and mRNA expression changes in human peripheral blood mononuclear cells (PBMC). A total of 12 teenagers with a substantial reduction in smoke quantity and a decrease in urine cotinine/creatinine ratio were enrolled in genomic analyses. In Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA), differentially expressed genes altered by smoke reduction were mainly associated with glucocorticoid receptor signaling pathway. The integrative analysis of microRNA and mRNA found eleven differentially expressed microRNAs negatively correlated with predicted target genes. CD83 molecule regulated by miR-4498 in human PBMC, was critical for the canonical pathway of communication between innate and adaptive immune cells. Our data demonstrated that microRNAs could regulate immune responses in human PBMC after habitual smokers quit smoking and support the potential translational value of microRNAs in regulating disease-relevant gene expression caused by tobacco smoke.

Advances in molecular biomarkers for gastric cancer: miRNAs as emerging novel cancer markers.

Carcinoma of the stomach is one of the most prevalent cancer types in the world. Although the incidence of gastric cancer is declining, the outcomes of gastric cancer patients remain dismal because of the lack of effective biomarkers to detect early gastric cancer. Modern biomedical research has explored many potential gastric cancer biomarker genes by utilising serum protein antigens, oncogenic genes or gene families through improving molecular biological technologies, such as microarray, RNA-Seq and the like. Recently, the small noncoding microRNAs (miRNAs) have been suggested to be critical regulators in the oncogenesis pathways and to serve as useful clinical biomarkers. This new class of biomarkers is emerging as a novel molecule for cancer diagnosis and prognosis, including gastric cancer. By translational suppression of target genes, miRNAs play a significant role in the gastric cancer cell physiology and tumour progression. There are potential implications of previously discovered gastric cancer molecular biomarkers and their expression modulations by respective miRNAs. Therefore, many miRNAs are found to play oncogenic roles or tumour-suppressing functions in human cancers. With the surprising stability of miRNAs in tissues, serum or other body fluids, miRNAs have emerged as a new type of cancer biomarker with immeasurable clinical potential.

Housekeeping protein-coding genes interrogated with tissue and individual variations.

Housekeeping protein-coding genes are stably expressed genes in cells and tissues that are thought to be engaged in fundamental cellular biological functions. They are often utilized as normalization references in molecular biology research and are especially important in integrated bioinformatic investigations. Prior studies have examined human housekeeping protein-coding genes by analyzing various gene expression datasets. The inclusion of different tissue types significantly impacted the discovery of housekeeping genes. In this report, we investigated particularly individual human subject expression differences in protein-coding genes across different tissue types. We used GTEx V8 gene expression datasets obtained from more than 16,000 human normal tissue samples. Furthermore, the Gini index is utilized to investigate the expression variations of protein-coding genes between tissue and individual donor subjects. Housekeeping protein-coding genes found using Gini index profiles may vary depending on the tissue subtypes investigated, particularly given the diverse sample size collections across the GTEx tissue subtypes. We subsequently selected major tissues and identified subsets of housekeeping genes with stable expression levels among human donors within those tissues. In this work, we provide alternative sets of housekeeping protein-coding genes that show more consistent expression patterns in human subjects across major solid organs. Weblink: https://hpsv.ibms.sinica.edu.tw .

Neutrophils Recruited by NKX2-1 Suppression via Activation of CXCLs/CXCR2 Axis Promote Lung Adenocarcinoma Progression.

NK2 Homeobox 1 (NKX2-1) is a well-characterized pathological marker that delineates lung adenocarcinoma (LUAD) progression. The advancement of LUAD is influenced by the immune tumor microenvironment through paracrine signaling. However, the involvement of NKX2-1 in modeling the tumor immune microenvironment is still unclear. Here, the downregulation of NKX2-1 is observed in high-grade LUAD. Meanwhile, single-cell RNA sequencing and Visium in situ capturing profiling revealed the recruitment and infiltration of neutrophils in orthotopic syngeneic tumors exhibiting strong cell-cell communication through the activation of CXCLs/CXCR2 signaling. The depletion of NKX2-1 triggered the expression and secretion of CXCL1, CXCL2, CXCL3, and CXCL5 in LUAD cells. Chemokine secretion is analyzed by chemokine array and validated by qRT-PCR. ATAC-seq revealed the restrictive regulation of NKX2-1 on the promoters of CXCL1, CXCL2, and CXCL5 genes. This phenomenon led to increased tumor growth, and conversely, tumor growth decreased when inhibited by the CXCR2 antagonist SB225002. This study unveils how NKX2-1 modulates the infiltration of tumor-promoting neutrophils by inhibiting CXCLs/CXCR2-dependent mechanisms. Hence, targeting CXCR2 in NKX2-1-low tumors is a potential antitumor therapy that may improve LUAD patient outcomes.

Co-modulated behavior and effects of differentially expressed miRNA in colorectal cancer.

BACKGROUND: MicroRNAs (miRNAs) are short noncoding RNAs (approximately 22 nucleotides in length) that play important roles in colorectal cancer (CRC) progression through silencing gene expression. Numerous dysregulated miRNAs simultaneously participate in the process of colon cancer development. However, the detailed mechanisms and biological functions of co-expressed miRNA in colorectal carcinogenesis have yet to be fully elucidated. RESULTS: The objective of this study was to identify the dysfunctional miRNAs and their target mRNAs using a wet-lab experimental and dry-lab bioinformatics approach. The differentially expressed miRNA candidates were identified from 2 miRNA profiles, and were confirmed in CRC clinical samples using reported target genes of dysfunctional miRNAs to perform functional pathway enrichment analysis. Potential target gene candidates were predicted by an in silico search, and their expression levels between normal and colorectal tumor tissues were further analyzed using real-time polymerase chain reaction (RT-PCR). CONCLUSION: Fifteen dysfunctional miRNAs were engaged in metastasis-associated pathways through comodulating 7 target genes, which were identified by using a multi-step approach. The roles of these candidate genes are worth further exploration in the progression of colon cancer, and could potentially be targets in future therapy.

dbDNV: a resource of duplicated gene nucleotide variants in human genome.

Gene duplications are scattered widely throughout the human genome. A single-base difference located in nearly identical duplicated segments may be misjudged as a single nucleotide polymorphism (SNP) from individuals. This imperfection is undistinguishable in current genotyping methods. As the next-generation sequencing technologies become more popular for sequence-based association studies, numerous ambiguous SNPs are rapidly accumulated. Thus, analyzing duplication variations in the reference genome to assist in preventing false positive SNPs is imperative. We have identified >10% of human genes associated with duplicated gene loci (DGL). Through meticulous sequence alignments of DGL, we systematically designated 1,236,956 variations as duplicated gene nucleotide variants (DNVs). The DNV database (dbDNV) (http://goods.ibms.sinica.edu.tw/DNVs/) has been established to promote more accurate variation annotation. Aside from the flat file download, users can explore the gene-related duplications and the associated DNVs by DGL and DNV searches, respectively. In addition, the dbDNV contains 304,110 DNV-coupled SNPs. From DNV-coupled SNP search, users observe which SNP records are also variants among duplicates. This is useful while ∼58% of exonic SNPs in DGL are DNV-coupled. Because of high accumulation of ambiguous SNPs, we suggest that annotating SNPs with DNVs possibilities should improve association studies of these variants with human diseases.

A unified framework of overlapping genes: towards the origination and endogenic regulation.

Overlapping genes are pairs of adjacent genes whose genomic regions partially overlap. They are notable by their potential intricate regulation, such as cis-regulation of nested gene-promoter configurations, and post-transcriptional regulation of natural antisense transcripts. The originations and consequent detailed regulation remain obscure. Herein, we propose a unified framework comprising biological classification rules followed by extensive analyses, namely, exon-sharing analysis, a human-mouse conservation study, and transcriptome analysis of hundreds of microarrays and transcriptome sequencing data (mRNA-Seq). We demonstrate that the tail-to-tail architecture would result from sharing functional elements in 3'-untranslated regions (3'-UTRs) of pre-existing genes. Dissimilarly, we illustrate that the other gene overlaps would originate from a new gene arising in a pre-existing gene locus. Interestingly, these types of coupled overlapping genes may influence each other synergistically or competitively during transcription, depending on the promoter configurations. This framework discloses distinctive characteristics of overlapping genes to be a foundation for a further comprehensive understanding of them.

VIP DB--a viral protein domain usage and distribution database.

During the viral infection and replication processes, viral proteins are highly regulated and may interact with host proteins. However, the functions and interaction partners of many viral proteins have yet to be explored. Here, we compiled a VIral Protein domain DataBase (VIP DB) to associate viral proteins with putative functions and interaction partners. We systematically assign domains and infer the functions of proteins and their protein interaction partners from their domain annotations. A total of 2,322 unique domains that were identified from 2,404 viruses are used as a starting point to correlate GO classification, KEGG metabolic pathway annotation and domain-domain interactions. Of the unique domains, 42.7% have GO records, 39.6% have at least one domain-domain interaction record and 26.3% can also be found in either mammals or plants. This database provides a resource to help virologists identify potential roles for viral protein. All of the information is available at http://vipdb.cgu.edu.tw.

MetaMirClust: discovery of miRNA cluster patterns using a data-mining approach.

Recent genome-wide surveys on ncRNA have revealed that a substantial fraction of miRNA genes is likely to form clusters. However, the evolutionary and biological function implications of clustered miRNAs are still elusive. After identifying clustered miRNA genes under different maximum inter-miRNA distances (MIDs), this study intended to reveal evolution conservation patterns among these clustered miRNA genes in metazoan species using a computation algorithm. As examples, a total of 15-35% of known and predicted miRNA genes in nine selected species constitute clusters under the MIDs ranging from 1kb to 50kb. Intriguingly, 33 out of 37 metazoan miRNA clusters in 56 metazoan genomes are co-conserved with their up/down-stream adjacent protein-coding genes. Meanwhile, a co-expression pattern of miR-1 and miR-133a in the mir-133-1 cluster has been experimentally demonstrated. Therefore, the MetaMirClust database provides a useful bioinformatic resource for biologists to facilitate the advanced interrogations on the composition of miRNA clusters and their evolution patterns.

Aberrant expression of miR-196a in gastric cancers and correlation with recurrence.

MicroRNAs (miRNAs) are short noncoding RNAs (~22 nt) that play important roles in the pathogenesis of human diseases by negatively regulating gene expression. Here, we examined the relationship between miR-196a and gastric cancer.By the analysis of 72 gastric cancer samples, we found that the expression level of miR-196a microRNA significantly increased in primary gastric cancer tissues versus adjacent normal tissues. In addition, extracellular miR-196a detected in conditioned medium was strongly correlated with its cellular expression status and increased circulating miR-196a in patient serum was associated with gastric cancer disease status and relapse. Furthermore, ectopic expression of miR-196a microRNA promoted the epithelial-mesenchymal transition and migration/invasion capabilities of transfected cells, suggesting its oncogenic potential in gastric cancer progression. Altogether, our data demonstrate that miR-196a exerts an oncogenic role in gastric cancer and miR-196a may be a novel biomarker for detecting gastric cancer and for monitoring disease recurrence.

Transcriptional regulation of miR-196b by ETS2 in gastric cancer cells.

E26 transformation-specific sequence (ETS)-2 is a transcriptional modulator located on chromosome 21, alterations in its expression have been implicated with a reduced incidence of solid tumors in Down syndrome patients. MicroRNAs (miRNAs) are thought to participate in diverse biological functions; however, the regulation of miRNAs is not well characterized. Recently, we reported that miR-196b is highly expressed in gastric cancers. Herein, we demonstrate that miR-196b expression was significantly repressed by ETS2 during gastric cancer oncogenesis. We demonstrate that knockdown of endogenous ETS2 expression increases miR-196b expression. A genomic region between -751 and -824 bp upstream of the miR-196b transcriptional start site was found to be critical for the repression activity. This putative regulatory promoter region contains three potential ETS2-binding motifs. Mutations within the ETS2 binding sites blocked the repression activity of ETS2. Furthermore, knockdown of ETS2 or overexpression of miR-196b significantly induced migration and invasion in gastric cancer cells. In addition, alterations in ETS2 and miR-196b expression in gastric cancer cell lines affected the expression of epithelial-mesenchymal transition-related genes. The levels of vimentin, matrix metalloproteinase (MMP)-2 and MMP9 were drastically induced, but levels of E-cadherin were decreased in shETS2- or miR-196b-transfected cells. Our data indicate that ETS2 plays a key role in controlling the expression of miR-196b, and miR-196b may mediate the tumor suppressor effects of ETS2. We demonstrated that miR-196b was transcriptionally regulated by ETS2 and there was an inverse expression profile between miR-196b and ETS2 in clinical samples. This finding could be beneficial for the development of effective cancer diagnostic and alternative therapeutic strategies.

Identification of gene-oriented exon orthology between human and mouse.

BACKGROUND: Gene orthology has been well studied in the evolutionary area and is thought to be an important implication to functional genome annotations. As the accumulation of transcriptomic data, alternative splicing is taken into account in the assignments of gene orthologs and the orthology is suggested to be further considered at transcript level. Whether gene or transcript orthology, exons are the basic units that represent the whole gene structure; however, there is no any reported study on how to build exon level orthology in a whole genome scale. Therefore, it is essential to establish a gene-oriented exon orthology dataset. RESULTS: Using a customized pipeline, we first build exon orthologous relationships from assigned gene orthologs pairs in two well-annotated genomes: human and mouse. More than 92% of non-overlapping exons have at least one ortholog between human and mouse and only a small portion of them own more than one ortholog. The exons located in the coding region are more conserved in terms of finding their ortholog counterparts. Within the untranslated region, the 5' UTR seems to have more diversity than the 3' UTR according to exon orthology designations. Interestingly, most exons located in the coding region are also conserved in length but this conservation phenomenon dramatically drops down in untranslated regions. In addition, we allowed multiple assignments in exon orthologs and a subset of exons with possible fusion/split events were defined here after a thorough analysis procedure. CONCLUSIONS: Identification of orthologs at the exon level is essential to provide a detailed way to interrogate gene orthology and splicing analysis. It could be used to extend the genome annotation as well. Besides examining the one-to-one orthologous relationship, we manage the one-to-multi exon pairs to represent complicated exon generation behavior. Our results can be further applied in many research fields studying intron-exon structure and alternative/constitutive exons in functional genomic areas.

miRNA arm selection and isomiR distribution in gastric cancer.

BACKGROUND: MicroRNAs (miRNAs) are small non-protein-coding RNAs. miRNA genes need several biogenesis steps to form function miRNAs. However, the precise mechanism and biology involved in the mature miRNA molecules are not clearly investigated. In this study, we conducted in-depth analyses to examine the arm selection and isomiRs using NGS platform. METHODS: We sequenced small RNAs from one pair of normal and gastric tumor tissues with Solexa platform. By analyzing the NGS data, we quantified the expression profiles of miRNAs and isomiRs in gastric tissues. Then, we measured the expression ratios of 5p arm to 3p arm of the same pre-miRNAs. And, we used Kolmogorov-Smirnov (KS) test to examine isomiR pattern difference between tissues. RESULTS: Our result showed the 5p arm and 3p arm miRNA derived from the same pre-miRNAs have different tissue expression preference, one preferred normal tissue and the other preferred tumor tissue, which strongly implied that there could be other mechanism controlling mature miRNA selection in addition to the known hydrogen-bonding selection rule. Furthermore, by using the KS test, we demonstrated that some isomiR types preferentially occur in normal gastric tissue but other types prefer tumor gastric tissue. CONCLUSIONS: Arm selections and isomiR patterns are significantly varied in human cancers by using deep sequencing NGS data. Our results provided a novel research topic in miRNA regulation study. With advanced bioinformatics and molecular biology studies, more robust conclusions and insight into miRNA regulation can be achieved in the near future.

Meta-analytical biomarker search of EST expression data reveals three differentially expressed candidates.

BACKGROUND: Researches have been conducted for the identification of differentially expressed genes (DEGs) by generating and mining of cDNA expressed sequence tags (ESTs) for more than a decade. Although the availability of public databases make possible the comprehensive mining of DEGs among the ESTs from multiple tissue types, existing studies usually employed statistics suitable only for two categories. Multi-class test has been developed to enable the finding of tissue specific genes, but subsequent search for cancer genes involves separate two-category test only on the ESTs of the tissue of interest. This constricts the amount of data used. On the other hand, simple pooling of cancer and normal genes from multiple tissue types runs the risk of Simpson's paradox. Here we presented a different approach which searched for multi-cancer DEG candidates by analyzing all pertinent ESTs in all categories and narrowing down the cancer biomarker candidates via integrative analysis with microarray data and selection of secretory and membrane protein genes as well as incorporation of network analysis. Finally, the differential expression patterns of three selected cancer biomarker candidates were confirmed by real-time qPCR analysis. RESULTS: Seven hundred and twenty three primary DEG candidates (p-value < 0.05 and lower bound of confidence interval of odds ratio ≥ 1.65) were selected from a curated EST database with the application of Cochran-Mantel-Haenszel statistic (CMH). GeneGO analysis results indicated this set as neoplasm enriched. Cross-examination with microarray data further narrowed the list down to 235 genes, among which 96 had membrane or secretory annotations. After examined the candidates in protein interaction network, public tissue expression databases, and literatures, we selected three genes for further evaluation by real-time qPCR with eight major normal and cancer tissues. The higher-than-normal tissue expression of COL3A1, DLG3, and RNF43 in some of the cancer tissues is in agreement with our in silico predictions. CONCLUSIONS: Searching digitized transcriptome using CMH enabled us to identify multi-cancer differentially expressed gene candidates. Our methodology demonstrated simultaneously analysis for cancer biomarkers of multiple tissue types with the EST data. With the revived interest in digitizing the transcriptomes by NGS, cancer biomarkers could be more precisely detected from the ESTs. The three candidates identified in this study, COL3A1, DLG3, and RNF43, are valuable targets for further evaluation with a larger sample size of normal and cancer tissue or serum samples.