A global transcriptome analysis of a dog model of congestive heart failure with the human genome as a reference.

BACKGROUND: The global molecular changes in cardiac tissue during congestive heart failure (CHF) have not been fully examined. Transcriptome analysis with the use of next-generation sequencers is a useful tool for elucidating the pathogenesis of CHF. Although there are some advantages in a dog CHF model, transcriptome analyses in dogs are limited by the relative lack of genomic information. METHODS AND RESULTS: The transcriptome analysis of hearts from dogs with CHF was conducted with the use of a genome analyzer and the Casava software. The mRNA sequence reads showed alignments with ∼800 of 1,019 genes from the dog reference database. On the other hand, the reads aligned with ∼15,000 of the 21,407 genes in the hg19 human reference database. The correlation of expressed genes was extremely high (r = 0.93; P < .0001) between the dog and human databases. A pathway analysis using the hg19 reference revealed increased expression of p53 pathway-related (P < 10(-10)) and inflammatory interleukin-related (P < 10(-10)) genes in the CHF model. CONCLUSIONS: The use of the human genome as a reference in global transcriptome analyses of dogs is a useful approach for investigating diseases such as CHF. Such an approach would also be useful for analyzing disease models in other experimental animals.

Transcriptome analysis of a dog model of congestive heart failure shows that collagen-related 2-oxoglutarate-dependent dioxygenases contribute to heart failure.

Fibrosis is an important pathological mechanism in heart failure (HF) and is associated with poor prognosis. We analyzed fibrosis in HF patients using transcriptomic data. Genes differentially expressed between normal control and congestive HF (CHF) dogs included P3H1, P3H2, P3H4, P4HA2, PLOD1 and PLOD3, which belong to the 2-oxoglutarate-dependent dioxygenases (2OGD) superfamily that stabilizes collagen during fibrosis. Quantitative polymerase chain reaction analysis demonstrated 2OGD gene expression was increased in CHF samples compared with normal left ventricle (LV) samples. 2OGD gene expression was repressed in angiotensin converting enzyme inhibitor-treated samples. These genes, activated the hydroxylation of proline or lysin residues of procollagen mediated by 2-oxoglutaric acid and O2, produce succinic acid and CO2. Metabolic analysis demonstrated the concentration of succinic acid was significantly increased in CHF samples compared with normal LV samples. Fibrosis was induced in human cardiac fibroblasts by TGF-ß1 treatment. After treatment, the gene and protein expressions of 2OGD, the concentration of succinic acid, and the oxygen consumption rate were increased compared with no treatment. This is the first study to show that collagen-related 2OGD genes contribute to HF during the induction of fibrosis and might be potential therapeutic targets for fibrosis and HF.

Analysis of the characteristics of chemotherapy-resistant renal cell carcinomas based on global transcriptional analysis of their tissues and cell lines.

Starvation-resistant renal cell carcinoma (RCC) cell lines are considered dormant-state cells that survive even under glucose starvation. The cellular biological and global transcriptional analysis using these cells identified potential markers of chemotherapy-resistant RCC and therapeutic agent candidates. Recently, we showed that ARL4C was a predictive biomarker for poor prognosis in patients with chemotherapy-resistant RCC by the global transcriptional analysis of patient primary tissues. The objective of this study was to identify the characteristics of chemotherapy-resistant RCC by the global transcriptional analysis of primary tissues of patients with RCC and RCC cell lines. The connective global transcriptional analysis showed that two starvation-resistant RCC cell lines, SW839 and KMRC-1, were strongly correlated to tissues of patients with chemotherapy-resistant RCC and showed high expressions of invasive- and proliferation-related genes. We found fibronectin (FN1) expression was a predictive biomarker in some patients with chemotherapy-resistant RCC, which especially correlated with two starvation-resistant RCC cell lines. These results indicate these cell lines emulate chemotherapy-resistant RCC and might be useful in the search for markers to predict poor prognosis and in the development of therapeutic agents and their index markers for chemotherapy-resistant RCCs.

ADP-ribosylation factor-like 4C is a predictive biomarker of poor prognosis in patients with renal cell carcinoma.

Renal cell carcinoma (RCC) has the high mortality rate among urological malignancies. The development of RCC cannot be effectively reduced by molecular targeted therapies based on nutrient deprivation, such as inhibition of tumor angiogenesis. The objective of this study was to identify predictive biomarkers of poor prognosis and therapeutic molecular targets in patients with RCC. Two independent cohorts were analyzed in the present study. Global transcriptomics were used in the first cohort (43 patients with RCC) to identify biomarker genes. Each identified biomarker was subsequently analyzed using immunohistochemistry in the second cohort (97 patients with RCC). Following transcriptomics, biomarkers were evaluated using receiver operating characteristic curve analysis. Predictive accuracy for poor survivals was assessed using the log-rank test and Cox multivariate analysis. Global transcriptomic analysis in the first cohort focusing on cases with survival periods <2 years after initial diagnosis of metastasis detected seven overexpressed genes, which correlated with poor prognosis. The ADP-ribosylation factor-like 4C (ARL4C) exhibited the best accuracy in the receiver operating characteristic curve analysis and predicted poor survival in the first cohort (log-rank test, P<0.001; Cox multivariate analysis, hazard ratio =167, P=0.005). In the second cohort, the expression of ARL4C was semi-quantitatively evaluated through immunohistochemistry. Twenty-seven cases showed high levels of ARL4C, confirming a significant association with shorter survivals (log-rank test, P<0.001; Cox multivariate analysis, hazard ratio =9.41, P=0.004). ARL4C was shown to be a predictive biomarker for poor prognosis in patients with RCC and may be a novel target in the treatment of RCC.

Hydroxyl-HIF2-alpha is potential therapeutic target for renal cell carcinomas.

Dormant cancer cells are deprivation-resistant, and cause a number of problems for therapeutic approaches for cancers. Renal cell carcinomas (RCCs) include deprivation-resistant cells that are resistant to various treatments. In this study, the specific characteristics of deprivation-resistant cells were transcriptionally identified by next generation sequencing. The hypoxia-inducible factors (HIF) transcription factor network was significantly enhanced in deprivation-resistant RCCs compared to the sensitive RCCs. Deprivation-resistant RCCs, that had lost Von Hippel-Lindau tumor suppressor expression, expressed hydroxyl-HIF2-alpha in the nucleus, but not sensitive-RCCs. Hydroxyl-HIF-alpha was also expressed in nuclei of RCC tissue samples. Knockdown for HIF2-alpha, but not HIF1-alpha, induced cell death related to a reduction in HIF-related gene expression in deprivation-resistant RCC cells. Chetomin, a nuclear HIF-inhibitor, induced marked level of cytotoxicity in deprivation-resistant cells, similar to the knockdown of HIF2-alpha. Therefore, hydroxyl-HIF2-alpha might be a potential therapeutic target for RCCs.

A novel large deletion (exons 12, 13) and a missense mutation (p.G46R) in the PAH in a Japanese patient with phenylketonuria.

BACKGROUND: Phenylketonuria (PKU) is caused by a defect in phenylalanine hydroxylase (PAH). More than 500 mutations have been reported for the gene encoding PAH. However, approximately 1%-5% of these include large deletions and large duplications that cannot be detected by conventional methods. METHODS: In this report we tried to fully characterize a PAH-deficient patient. The patient was a 2-year-old Japanese boy who was diagnosed with classical PKU at the time of neonatal screening, which was confirmed by the tetrahydrobiopterin-loading test. PCR-related direct sequencing and multiplex ligation-dependent probe amplification (MLPA) were used to analyze of the PAH of the patient. RESULTS: Using PCR-related direct sequencing method, we could detect only a heterozygous novel missense mutation: p.136G>C (p.G46R). A second mutation was detected by MLPA. The patient was heterozygous for a novel large deletion of exons 12 and 13: c.1200-?_1359+?del (EX12_13del). For genetic counseling, an accurate genetic diagnosis is often necessary. CONCLUSIONS: Through a combination of MLPA and conventional methods, the success rate of PAH mutation identification can be close to 100%.

Analysis of new biomarkers for cholangiocarcinoma.

Cholangiocarcinoma is one of the most serious diseases in northeast Thailand, where its incidence is reported to be the highest in the world. We tried to develop a new method to detect cholangiocarcinoma in the early stages using serum proteins. We found that after fluorescent labeling of the sugar moiety of serum proteins, a new peak was identified, which might be a promising marker for cholangiocarcinoma.

Study of global transcriptional changes of N-GlcNAc2 proteins-producing T24 bladder carcinoma cells under glucose deprivation.

Increased levels of N-linked (ß-N- acetylglucosamine)2 [N-GlcNAc2]-modified proteins have been recognized to be an effective response to glucose deprivation. In the first step of this study, using a next generation sequencer, we investigated the global transcriptional changes induced by glucose deprivation in a T24 bladder carcinoma cell line, producing N-GlcNAc2-modified proteins under glucose deprivation. Our transcriptome analysis revealed significant up-regulation of the UDP-GlcNAc biosynthesis pathway and unfolded protein response genes, and down-regulation of G2/M transition-related genes containing mitotic kinases. Our biological analysis confirmed that N-GlcNAc2-modified proteins were localized with BiP proteins in the ER. G2/M arrest was caused by glucose deprivation in T24 cells. Moreover, the knockdown of unfolded protein response genes induced the expressional recovery of mitotic kinases under glucose deprivation. Taken together, our results suggest N-GlcNAc2-modified proteins produced under glucose deprivation caused unfolded protein response in the ER, and that this response induced G2/M arrest.