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Background: Although much research effort has been devoted to elucidating lung cancer, the molecular mechanism of tumorigenesis still remains unclear. A major challenge to improve the understanding of lung cancer is the difficulty of identifying reproducible differentially expressed genes across independent studies, due to their low consistency. To enhance the reproducibility of the findings, an integrated analysis was performed to identify regulatory SNPs. Thirty-two pairs of tumor and adjacent normal lung tissue specimens were analyzed using Affymetrix U133plus2.0, Affymetrix SNP 6.0, and Illumina Infinium Methylation microarrays. Copy number variations (CNVs) and methylation alterations were analyzed and paired t-tests were used to identify differentially expressed genes. Results: A total of 505 differentially expressed genes were identified, and their dysregulated patterns moderately correlated with CNVs and methylation alterations based on the hierarchical clustering analysis. Subsequently, three statistical approaches were performed to explore regulatory SNPs, which revealed that the genotypes of 551 and 66 SNPs were associated with CNV and changes in methylation, respectively. Among them, downstream transcriptional dysregulation was observed in 9 SNPs for CNVs and 4 SNPs for methylation alterations. Conclusions: In summary, these identified SNPs concurrently showed the same direction of gene expression changes with genetic modifications, suggesting their pivotal roles in the genome for non-smoking women with lung adenocarcinoma. [ABSTRACT FROM AUTHOR]

Background: MicroRNAs (miRNAs) are short, non-coding RNA molecules that play critical roles in human malignancy. However, the regulatory characteristics of miRNAs in triple-negative breast cancer, a phenotype of breast cancer that does not express the genes for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, are still poorly understood. Methods: In this study, miRNA expression profiles of 24 triple-negative breast cancers and 14 adjacent normal tissues were analyzed using deep sequencing technology. Expression levels of miRNA reads were normalized with the quantile-quantile scaling method. Deregulated miRNAs in triple-negative breast cancer were identified from the sequencing data using the Student's t-test. Quantitative reverse transcription PCR validations were carried out to examine miRNA expression levels. Potential target candidates of a miRNA were predicted using published target prediction algorithms. Luciferase reporter assay experiments were performed to verify a putative miRNA-target relationship. Validated molecular targets of the deregulated miRNAs were retrieved from curated databases and their associations with cancer progression were discussed. Results: A novel 25-miRNA expression signature was found to effectively distinguish triple-negative breast cancers from surrounding normal tissues in a hierarchical clustering analysis. We documented the evidence of seven polycistronic miRNA clusters preferentially harboring deregulated miRNAs in triple-negative breast cancer. Two of these miRNA clusters (miR-143-145 at 5q32 and miR-497-195 at 17p13.1) were markedly down-regulated in triple-negative breast cancer, while the other five miRNA clusters (miR-17-92 at 13q31.3, miR-183-182 at 7q32.2, miR-200-429 at 1p36.33, miR-301b-130b at 22q11.21, and miR-532-502 at Xp11.23) were up-regulated in triplenegative breast cancer. Moreover, miR-130b-5p from the miR-301b-130b cluster was shown to directly repress the cyclin G2 (CCNG2) gene, a crucial cell cycle regulator, in triple-negative breast cancer cells. Luciferase reporter assays showed that miR-130b-5p-mediated repression of CCNG2 was dependent on the sequence of the 3'-untranslated region. The findings described in this study implicate a miR-130b-5p-CCNG2 axis that may be involved in the malignant progression of triple-negative breast cancer. Conclusions: Our work delivers a clear picture of the global miRNA regulatory characteristics in triple-negative breast cancer and extends the current knowledge of microRNA regulatory network. [ABSTRACT FROM AUTHOR]

A need for more accurate and reliable radiation dosimetry has become increasingly important due to the possibility of a large-scale radiation emergency resulting from terrorism or nuclear accidents. Although traditional approaches provide accurate measurements, such methods usually require tedious effort and at least two days to complete. Therefore, we provide a new method for rapid prediction of radiation exposure. Eleven microarray datasets were classified into two groups based on their radiation doses and utilized as the training samples. For the two groups, Student's t-tests and resampling tests were used to identify biomarkers, and their gene expression ratios were used to develop a prediction model. The performance of the model was evaluated in four independent datasets, and Ingenuity pathway analysis was performed to characterize the associated biological functions. Our meta-analysis identified 29 biomarkers, showing approximately 90% and 80% accuracy in the training and validation samples. Furthermore, the 29 genes significantly participated in the regulation of cell cycle, and 19 of them are regulated by three well-known radiation-modulated transcription factors: TP53, FOXM1 and ERBB2. In conclusion, this study demonstrates a reliable method for identifying biomarkers across independent studies and high and reproducible prediction accuracy was demonstrated in both internal and external datasets. [ABSTRACT FROM AUTHOR]

Hypoxia and reoxygenation are common characteristics of solid tumors, which lead to oxidative stress and activation of stress-response genes. Previously, we observed that N-myc downstream-regulated gene 1 (NDRG1) was strongly down-regulated after shifting to reoxygenation, but the regulatory mechanism of NDRG1 remained elusive. Here we focused on the regulation of NDRG1 by microRNAs (miRNAs). Breast cancer MCF-7 cells were cultured under hypoxia for 24 h followed by 24 h of reoxygenation. The miRNA profiles were examined by Nanostring nCounter assays. Forty-three miRNAs had significant changes upon reoxygenation. In silico analysis identified four oxygen-sensitive miRNAs whose seed regions perfectly matched the 3'-UTR of NDRG1. In particular, miR-769-3p was able to inhibit the expression of NDRG1, which caused a significant reduction of NDRG1 protein upon reoxygenation. Furthermore, overexpression of miR-769-3p significantly inhibited cell proliferation and enhanced apoptosis. Our results revealed that miR-769-3p can functionally regulate NDRG1 during changes in oxygen concentration. [ABSTRACT FROM AUTHOR]

The ability of adult stem cells to reside in a quiescent state is crucial for preventing premature exhaustion of the stem cell pool. However, the intrinsic epigenetic factors that regulate spermatogonial stem cell quiescence are largely unknown. Here, we investigate in micehowDNA methyltransferase 3-like (DNMT3L), an epigenetic regulator important for interpreting chromatin context and facilitating de novo DNA methylation, sustains the long-term male germ cell pool. We demonstrated that stem cell-enriched THY1+ spermatogonial stem/ progenitor cells (SPCs) constituted a DNMT3L-expressing population in postnatal testes. DNMT3L influenced the stability of promyelocytic leukemia zinc finger (PLZF), potentially by downregulating Cdk2/CDK2 expression, which sequestered CDK2-mediated PLZF degradation. Reduced PLZF in Dnmt3l KO THY1+ cells released its antagonist, Sallike protein 4A (SALL4A), which is associated with overactivated ERK and AKT signaling cascades. Furthermore, DNMT3L was required to suppress the cell proliferation-promoting factorSALL4BinTHY1+ SPCs and to prevent premature stemcell exhaustion. Our results indicate that DNMT3L is required to delicately balance the cycling and quiescence of SPCs. These findings reveal a novel role for DNMT3L in modulating postnatal SPC cell fate decisions. [ABSTRACT FROM AUTHOR]

Numerous efforts have been made to elucidate the etiology and improve the treatment of lung cancer, but the overall five-year survival rate is still only 15%. Although cigarette smoking is the primary risk factor for lung cancer, only 7% of female lung cancer patients in Taiwan have a history of smoking. Since cancer results from progressive accumulation of genetic aberrations, genomic rearrangements may be early events in carcinogenesis. Results In order to identify biomarkers of early-stage adenocarcinoma, the genome-wide DNA aberrations of 60 pairs of lung adenocarcinoma and adjacent normal lung tissue in nonsmoking women were examined using Affymetrix Genome-Wide Human SNP 6.0 arrays. Common copy number variation (CNV) regions were identified by ⩾30% of patients with copy number beyond 2 ± 0.5 of copy numbers for each single nucleotide polymorphism (SNP) and at least 100 continuous SNP variant loci. SNPs associated with lung adenocarcinoma were identified by McNemar's test. Loss of heterozygosity (LOH) SNPs were identified in ⩾18% of patients with LOH in the locus. Aberration of SNP rs10248565 at HDAC9 in chromosome 7p21.1 was identified from concurrent analyses of CNVs, SNPs, and LOH. Conclusion The results elucidate the genetic etiology of lung adenocarcinoma by demonstrating that SNP rs10248565 may be a potential biomarker of cancer susceptibility. [ABSTRACT FROM AUTHOR]

DNA methylation plays an important role in regulating cell growth and disease development. Methylation profiles are examined by bisulfite conversion; however, the lack of markers for bisulfite conversion efficiency and appropriate internal control genes remains a major challenge. To address these issues, we utilized two bioinformatics approaches, coefficients of variances and resampling tests, to identify probes showing stable methylation levels from several independent microarray datasets. Mass spectrometry validated the consistently high methylation levels of the five probes (N4BP2, EGFL8, CTRB1, TSPAN3, and ZNF690) in 13 human tissue types from 24 cell lines. Linear associations between detected methylation levels and methyl concentrations of DNA samples were further demonstrated in three genes (N4BP2,EGFL8, and CTRB1). To summarize, we identified five genes which may serve as internal controls for methylation studies by analyzing large-scale microarray data, and three of them can be used as markers for evaluating the efficiency of bisulfite conversion. [ABSTRACT FROM AUTHOR]

Background: Salamanders are unique among vertebrates in their ability to completely regenerate amputated limbs through the mediation of blastema cells located at the stump ends. This regeneration is nerve-dependent because blastema formation and regeneration does not occur after limb denervation. To obtain the genomic information of blastema tissues, de novo transcriptomes from both blastema tissues and denervated stump ends of Ambystoma mexicanum (axolotls) 14 days post-amputation were sequenced and compared using Solexa DNA sequencing. Results: The sequencing done for this study produced 40,688,892 reads that were assembled into 307,345 transcribed sequences. The N50 of transcribed sequence length was 562 bases. A similarity search with known proteins identified 39,200 different genes to be expressed during limb regeneration with a cut-off E-value exceeding 10-5. We annotated assembled sequences by using gene descriptions, gene ontology, and clusters of orthologous group terms. Targeted searches using these annotations showed that the majority of the genes were in the categories of essential metabolic pathways, transcription factors and conserved signaling pathways, and novel candidate genes for regenerative processes. We discovered and confirmed numerous sequences of the candidate genes by using quantitative polymerase chain reaction and in situ hybridization. Conclusion: The results of this study demonstrate that de novo transcriptome sequencing allows gene expression analysis in a species lacking genome information and provides the most comprehensive mRNA sequence resources for axolotls. The characterization of the axolotl transcriptome can help elucidate the molecular mechanisms underlying blastema formation during limb regeneration. [ABSTRACT FROM AUTHOR]

Discrepancies in the prognosis of triple negative breast cancer exist between Caucasian and Asian populations. Yet, the gene signature of triple negative breast cancer specifically for Asians has not become available. Therefore, the purpose of this study is to construct a prediction model for recurrence of triple negative breast cancer in Taiwanese patients. Whole genome expression profiling of breast cancers from 185 patients in Taiwan from 1995 to 2008 was performed, and the results were compared to the previously published literature to detect differences between Asian and Western patients. Pathway analysis and Cox proportional hazard models were applied to construct a prediction model for the recurrence of triple negative breast cancer. Hierarchical cluster analysis showed that triple negative breast cancers from different races were in separate sub-clusters but grouped in a bigger cluster. Two pathways, cAMP-mediated signaling and ephrin receptor signaling, were significantly associated with the recurrence of triple negative breast cancer. After using stepwise model selection from the combination of the initial filtered genes, we developed a prediction model based on the genes SLC22A23, PRKAG3, DPEP3, MORC2, GRB7, and FAM43A. The model had 91.7% accuracy, 81.8% sensitivity, and 94.6% specificity under leave-one-out support vector regression. In this study, we identified pathways related to triple negative breast cancer and developed a model to predict its recurrence. These results could be used for assisting with clinical prognosis and warrant further investigation into the possibility of targeted therapy of triple negative breast cancer in Taiwanese patients. [ABSTRACT FROM AUTHOR]

The article discusses research on the molecular signature of nonsmoking female lung cancer patients in Taiwan, with a particular focus on the semaphorin gene family. A paired t test was used to identify differentially expressed genes in tumor tissues of sixty pairs of lung tissue specimens and were validated by reverse transcriptase-polymerase chain reaction (PCR) and immunohistochemistry. Results showed that several semaphorin gene family members were potential tumor suppressors.

Background: About 30-50% of Hodgkin lymphomas (HLs) harbor the Epstein-Barr virus (EBV), but the impact of EBV infection on clinical outcomes has been unclear. EBV-encoded small RNAs (EBERs) are presented in all EBV-infected cells, but their functions are still less understood. Results: EBER1 was transfected into two HL cell lines, KMH2 and L428, and microarrays were used to screen for EBER1-induced changes. We found that EBER1 suppressed p21cip1/waf1 transcription in HL cell lines. In addition, positive regulators of p21cip1/waf1 transcription, such as p53, EGR1, and STAT1, were decreased. Suppression of p21cip1/waf1 in the EBER1+ HL cell lines was associated with increased resistance to histone deacetylase inhibitors or proteasome inhibitors, drugs known to cause apoptosis by increasing p21cip1/waf1 levels. On biopsy specimens, EBV+ HLs had weaker expression of both p21cip1/waf1 and active caspase 3. Clinically, suppression of p21cip1/waf1 in EBV+ HLs was associated with a worse 2-year disease-free survival rate (45% for EBV+ HLs vs. 77% for EBV-HLs, p = 0.002). Conclusion: Although the underlying mechanisms are still relatively unclear, EBER1 inhibits p21cip1/waf1 transcription and prevents apoptosis through down-regulation of p53, EGR1, and STAT1. The anti-apoptotic activity of EBER1 may be important in the rescue of Reed-Sternberg cells from drug-induced apoptosis and in the clinical behaviors of EBV+ HLs. [ABSTRACT FROM AUTHOR]

In cancer, gene silencing via hypermethylation is as common as genetic mutations in p53. Understanding the relationship between mutant p53 and hypermethylation of other tumor suppressor genes is essential when elucidate mechanisms of tumor development. In this study, two isogenic human B lymphoblast cell lines with different p53 status include TK6 containing wild-type p53 and WTK1 with mutant p53 were used and contrasted. Lower levels of p16ink4A protein were detected in WTK1 cells than in TK6 cells, which were accompanied by increased DNA (cytosine-5)-methyltransferase 1 ( DNMT1) gene expression as well as hypermethylation of the p16 ink4A promoter. siRNA experiments to transiently knock down wild-type p53 in TK6 cells resulted in increase of DNMT1 expression as well as decrease of p16ink4A protein. Conversely, siRNA knockdown of mutant p53 in WTK1 cells did not alter either DNMT1 or p16ink4A protein levels. Furthermore, loss of suppression function of mutant p53 to DNMT1 in WTK1 was caused by the attenuation of its binding ability to the DNMT1 promoter. In summary, we provide evidences to elucidate the relationship between mutant p53 and DNMT1. Our results indicate that mutant p53 loses its ability to suppress DNMT1 expression, and thus enhances methylation levels of the p16 ink4A promoter and subsequently down-regulates p16ink4A protein. [ABSTRACT FROM AUTHOR]

To assess DNA damage remaining in peripheral lymphocytes, 48 individuals were evaluated twice for lymphocyte micronucleus frequencies by the cytokinesis-blocking cytochalasin B (CBMN) analysis post relocation from radio-contaminated apartments after various periods of time. The frequencies of CBMN at the first evaluation were significantly higher than those at the second examination (Chang et al., 1999c). These individuals were categorized into three groups: those with cumulative exposure of >300 mSv (defined as high exposure, HDose), those with 100-300 mSv (MDose) and those with <100 mSv (LDose). Using the Poisson mixed-effect model (Little et al., 1996), the estimated mean CBMN frequencies (‰) for individuals in HDose, MDose and LDose exposure categories when they had only recently relocated were 21.8, 17.6 and 15.4, respectively. The estimated mean duration post relocation for the CBMN frequencies of these individuals to reduce to 10.2, the second CBMN frequency, on average, was 47.5, 37.2 and 28.3 months in the three exposure groups, respectively. The rates of change in CBMN frequencies were shown to be significantly higher in the HDose group than in the MDose and LDose groups. The results suggested a characteristic dose-dependent decline in the CBMN frequencies in the exposed population post cessation of chronic low-dose ionizing radiation exposure. [ABSTRACT FROM AUTHOR]

Treatment of primary effusion lymphoma cells latently infected by Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus-8 [HHV-8]) with agents such as 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a lytic viral replication cycle, with an ordered gene expression program. Initial studies of the KSHV expression program following TPA induction using viral microarrays yielded useful information concerning the viral expression program, but precise kinetic assignments for some genes remained unclear. Classically, late herpesvirus genes require viral DNA replication for maximal expression. We used cidofovir (CDV), a nucleotide-analogue KSHV DNA polymerase inhibitor, to dissect KSHV expression into two components: genes expressed without viral DNA replication and those requiring it. The expression of known immediate-early or early genes (e.g., open reading frames [ORFs] 50, K8 bZIP, and 57) serving lytic regulatory roles was relatively unaffected by the presence of CDV, while known late capsid and tegument structural genes (e.g., ORFs 25, 26, 64, and 67) were CDV sensitive. Latency-associated transcript ORF 73 was unaffected by the presence of TPA or CDV, suggesting that it was constitutively expressed. Expression of several viral cellular gene homologs, including K2 (vIL-6), ORF 72 (vCyclin), ORF 74 (vGPCR), and K9 (vIRF-1), was unaffected by the presence of CDV, while that of others, such as K4.1 (vMIP-III), K11.1 (vIRF-2), and K10.5 (LANA2, vIRF-3), was inhibited. The results distinguish KSHV genes whose full expression required viral DNA replication from those that did not require it, providing additional insights into KSHV replication and pathogenesis strategies and helping to show which viral cell homologs are expressed at particular times during the lytic process. [ABSTRACT FROM AUTHOR]

Rickettsia prowazekii, the causative agent of epidemic typhus, has been responsible for millions of human deaths. Madrid E is an attenuated strain of R. prowazekii, while Breinl is a virulent strain. The genomic DNA sequence of Madrid E has recently been published. To study the genomic variations between Madrid E (reference) and Breinl (test) DNAs, cohybridization experiments were performed on a DNA microarray containing all 834 protein-coding genes of Madrid E. Of the 834 genes assessed, 24 genes showed 1.5- to 2.0-fold increases in hybridization signals in Breinl DNA compared to Madrid E DNA, indicating the presence of genomic variations in ∼3% of the total genes. Eighteen of these 24 genes are predicted to be involved in different functions. Southern blot analysis of five genes, virB4, ftsK, rfbE, lpxA, and rpoH, suggested the presence of an additional paralog(s) in Breinl, which might be related to the observed increase in hybridization signals. Studies by real-time reverse transcription-PCR revealed an increase in expression of the above-mentioned five genes and five other genes. In addition to the elevated hybridization signals of 24 genes observed in the Breinl strain, one gene (rp084) showed only 1/10 the hybridization signal of Madrid E. Further analysis of this gene by PCR and sequencing revealed a large deletion flanking the whole rp084 gene and part of the rp083 gene in the virulent Breinl strain. The results of this first rickettsial DNA microarray may provide some important information for the elucidation of pathogenic mechanisms of R. prowazekii. [ABSTRACT FROM AUTHOR]