Hisakazu Iwama

The genome sequence and structure of rice chromosome 1

The rice species Oryza sativa is considered to be a model plant because of its small genome size, extensive genetic map, relative ease of transformation and synteny with other cereal crops. Here we report the essentially complete sequence of chromosome 1, the longest chromosome in the rice genome. We summarize characteristics of the chromosome structure and the biological insight gained from the sequence. The analysis of 43.3 megabases (Mb) of non-overlapping sequence reveals 6,756 protein coding genes, of which 3,161 show homology to proteins of Arabidopsis thaliana, another model plant. About 30% (2,073) of the genes have been functionally categorized. Rice chromosome 1 is (G + C)-rich, especially in its coding regions, and is characterized by several gene families that are dispersed or arranged in tandem repeats. Comparison with a draft sequence indicates the importance of a high-quality finished sequence.

The antidiabetic drug metformin inhibits gastric cancer cell proliferation in vitro and in vivo

Recent studies suggest that metformin, which is commonly used as an oral anti-hyperglycemic agent of the biguanide family, may reduce cancer risk and improve prognosis, but the mechanisms by which metformin affects various cancers, including gastric cancer, remains unknown. The goal of the present study was to evaluate the effects of metformin on human gastric cancer cell proliferation in vitro and in vivo and to study microRNAs (miRNA) associated with antitumor effect of metformin. We used MKN1, MKN45, and MKN74 human gastric cancer cell lines to study the effects of metformin on human gastric cancer cells. Athymic nude mice bearing xenograft tumors were treated with or without metformin. Tumor growth was recorded after 4 weeks, and the expression of cell-cycle-related proteins was determined. In addition, we used miRNA array tips to explore the differences among miRNAs in MKN74 cells bearing xenograft tumors treated with or without metformin in vitro and in vivo. Metformin inhibited the proliferation of MKN1, MKN45, and MKN74 in vitro. Metformin blocked the cell cycle in G0–G1 in vitro and in vivo. This blockade was accompanied by a strong decrease of G1 cyclins, especially in cyclin D1, cyclin-dependent kinase (Cdk) 4, Cdk6 and by a decrease in retinoblastoma protein (Rb) phosphorylation. In addition, metformin reduced the phosphorylation of epidermal growth factor receptor and insulin-like growth factor-1 receptor in vitro and in vivo. The miRNA expression was markedly altered with the treatment of metformin in vitro and in vivo. Various miRNAs altered by metformin also may contribute to tumor growth in vitro and in vivo. Mol Cancer Ther; 11(3); 549–60. ©2012 AACR.

Effect of the anti-diabetic drug metformin in hepatocellular carcinoma in vitro and in vivo.

Metformin is a commonly used oral anti-hyperglycemic agent of the biguanide family. Recent studies suggest that metformin may reduce cancer risk and improve prognosis. However, the antitumor mechanism of metformin in several types of cancers, including hepatocellular carcinoma (HCC), has not been elucidated. The goal of the present study was to evaluate the effects of metformin on HCC cell proliferation in vitro and in vivo, and to study microRNAs (miRNAs) associated with the antitumor effect of metformin in vitro. We used the cell lines Alex, HLE and Huh7, and normal hepatocytes to study the effects of metformin on human HCC cells. In an in vivo study, athymic nude mice bearing xenograft tumors were treated with metformin or left untreated. Tumor growth was recorded after 4 weeks, and the expression of cell cycle-related proteins was determined. Metformin inhibited the proliferation of Alex, HLE and Huh7 cells in vitro and in vivo. Metformin blocked the cell cycle in G0/G1 in vitro and in vivo. This blockade was accompanied by a strong decrease of G1 cyclins, especially cyclin D1, cyclin E and cyclin-dependent kinase 4 (Cdk4). In addition, microRNA (miRNA) expression was markedly altered by the treatment with metformin in vitro and in vivo. In addition, various miRNAs induced by metformin also may contribute to the suppression of tumor growth. Our results demonstrate that metformin inhibits the growth of HCC, possibly by inducing G1 cell cycle arrest through the alteration of microRNAs.

Epstein-Barr virus detection in kidney biopsy specimens correlates with glomerular mesangial injury

To determine the relationship between the detection of Epstein-Barr virus (EBV)-specific DNA and glomerular injury, 33 renal needle-biopsy specimens that had been formalin-fixed and paraffin-embedded were analyzed using polymerase chain reaction (PCR) with subsequent nonradioactive Southern blot technique. Light microscopic examination and immunofluorescence were also performed. In 30 of 33 renal biopsy specimens, the beta globin gene could be successfully amplified as integrity controls. These 30 patients consisted of 12 patients with immunoglobulin A nephropathy (IgAN), 10 patients with minor glomerular abnormalities, 6 patients with membranous nephropathy, and 2 patients with focal/segmental lesions. EBV was detected in 7 of 12 patients with IgAN (58%), 3 of 6 patients with membranous nephropathy (50%), 0 of 10 patients with minor glomerular abnormalities (0%), and 2 of 2 patients with focal/segmental lesions. EBV detection was not disease specific. The EBV detection ratio of the group with glomerular mesangial lesions (64%; 9 of 14 patients) was significantly greater than those without (19%; 3 of 16 patients; P < 0.012, chi-square test). The EBV detection ratio of the group with glomerular lesions (60%; 12 of 20 patients) was significantly greater than those without (0%; 0 of 10 patients; P < 0.0016, Fishers exact test), and the EBV detection ratio of the group with fibrinogen deposits observed in immunofluorescence (73%; 11 of 15 patients) was significantly greater than those without (7%; 1 of 15 patients; P < 0.0002, chi-square test). The EBV detection ratio of the group with immunoglobulin deposits (57%; 12 of 21 patients) was also significantly greater than those without (0%; 0 of 9 patients; P < 0.0040, Fishers exact test). These data suggest that EBV can damage the glomerular mesangium beyond disease units and be mediated by immunoglobulin in patients with various chronic glomerulonephritides.

Evola : Ortholog database of all human genes in H-InvDB with manual curation of phylogenetic trees

Orthologs are genes in different species that evolved from a common ancestral gene by speciation. Currently, with the rapid growth of transcriptome data of various species, more reliable orthology information is prerequisite for further studies. However, detection of orthologs could be erroneous if pairwise distance-based methods, such as reciprocal BLAST searches, are utilized. Thus, as a sub-database of H-InvDB, an integrated database of annotated human genes (http://h-invitational.jp/), we constructed a fully curated database of evolutionary features of human genes, called ‘Evola’. In the process of the ortholog detection, computational analysis based on conserved genome synteny and transcript sequence similarity was followed by manual curation by researchers examining phylogenetic trees. In total, 18 968 human genes have orthologs among 11 vertebrates (chimpanzee, mouse, cow, chicken, zebrafish, etc.), either computationally detected or manually curated orthologs. Evola provides amino acid sequence alignments and phylogenetic trees of orthologs and homologs. In ‘dN/dS view’, natural selection on genes can be analyzed between human and other species. In ‘Locus maps’, all transcript variants and their exon/intron structures can be compared among orthologous gene loci. We expect the Evola to serve as a comprehensive and reliable database to be utilized in comparative analyses for obtaining new knowledge about human genes. Evola is available at http://www.h-invitational.jp/evola/.

Metformin Suppresses Expression of the Selenoprotein P Gene via an AMP-activated Kinase (AMPK)/FoxO3a Pathway in H4IIEC3 Hepatocytes

Background: The suppression of selenoprotein P production may be a novel therapeutic target for reducing insulin resistance. Results: Selenoprotein P expression was suppressed by metformin treatment, but co-administration of AMPK inhibitor or FoxO3a siRNA cancelled this suppression. Conclusion: Metformin suppresses selenoprotein P expression via the AMPK/FoxO3a pathway. Significance: The AMPK/FoxO3a pathway in the liver may be a therapeutic target for type 2 diabetes. Selenoprotein P (SeP; encoded by SEPP1 in humans) is a liver-derived secretory protein that induces insulin resistance in type 2 diabetes. Suppression of SeP might provide a novel therapeutic approach to treating type 2 diabetes, but few drugs that inhibit SEPP1 expression in hepatocytes have been identified to date. The present findings demonstrate that metformin suppresses SEPP1 expression by activating AMP-activated kinase (AMPK) and subsequently inactivating FoxO3a in H4IIEC3 hepatocytes. Treatment with metformin reduced SEPP1 promoter activity in a concentration- and time-dependent manner; this effect was cancelled by co-administration of an AMPK inhibitor. Metformin also suppressed Sepp1 gene expression in the liver of mice. Computational analysis of transcription factor binding sites conserved among the species resulted in identification of the FoxO-binding site in the metformin-response element of the SEPP1 promoter. A luciferase reporter assay showed that metformin suppresses Forkhead-response element activity, and a ChIP assay revealed that metformin decreases binding of FoxO3a, a direct target of AMPK, to the SEPP1 promoter. Transfection with siRNAs for Foxo3a, but not for Foxo1, cancelled metformin-induced luciferase activity suppression of the metformin-response element of the SEPP1 promoter. The overexpression of FoxO3a stimulated SEPP1 promoter activity and rescued the suppressive effect of metformin. Metformin did not affect FoxO3a expression, but it increased its phosphorylation and decreased its nuclear localization. These data provide a novel mechanism of action for metformin involving improvement of systemic insulin sensitivity through the regulation of SeP production and suggest an additional approach to the development of anti-diabetic drugs.

Human MicroRNAs Originated from Two Periods at Accelerated Rates in Mammalian Evolution

MicroRNAs (miRNAs) are short, noncoding RNAs that modulate genes posttranscriptionally. Frequent gains and losses of miRNA genes have been reported to occur during evolution. However, little is known systematically about the periods of evolutionary origin of the present miRNA gene repertoire of an extant mammalian species. Thus, in this study, we estimated the evolutionary periods during which each of 1,433 present human miRNA genes originated within 15 periods, from human to platypus–human common ancestral branch and a class “conserved beyond theria,” primarily using multiple genome alignments of 38 species, plus the pairwise genome alignments of five species. The results showed two peak periods in which the human miRNA genes originated at significantly accelerated rates. The most accelerated rate appeared in the period of the initial phase of hominoid lineage, and the second appeared shortly before Laurasiatherian divergence. Approximately 53% of the present human miRNA genes have originated within the simian lineage to human. In particular, approximately 28% originated within the hominoid lineage. The early phase of placental mammal radiation comprises approximately 28%, while no more than 15% of human miRNAs have been conserved beyond placental mammals. We also clearly showed a general trend, in which the miRNA expression level decreases as the miRNA becomes younger. Intriguingly, amid this decreasing trend of expression, we found one significant rise in the expression level that corresponded to the initial phase of the hominoid lineage, suggesting that increased functional acquisitions of miRNAs originated at this particular period.

Antitumor effect of metformin in esophageal cancer: In vitro study

Recent studies suggest that metformin, which is a member of the biguanide family and commonly used as an oral anti-hyperglycemic agent, may reduce cancer risk and improve prognosis of numerous types of cancer. However, the mechanisms underlying the antitumor effect of metformin on esophageal cancer remain unknown. The goal of the present study was to evaluate the effects of metformin on the proliferation of human ESCC in vitro, and to study changes in the expression profile of microRNAs (miRNAs), since miRNAs have previously been associated with the antitumor effects of metformin in other human cancers. The human ESCC cell lines T.T, KYSE30 and KYSE70 were used to study the effects of metformin on human ESCC in vitro. In addition, we used miRNA array tips to explore the differences between miRNAs in KYSE30 cells with and without metformin treatment. Metformin inhibited the proliferation of T.T, KYSE30 and KYSE70 cells in vitro. Metformin blocked the cell cycle in G0/G1 in vitro. This blockade was accompanied by a strong decrease of G1 cyclins, especially cyclin D1, as well as decreases in cyclin-dependent kinase (Cdk)4, Cdk6 and phosphorylated retinoblastoma protein (Rb). In addition, the expression of miRNAs was markedly altered with the treatment of metformin in vitro. Metformin inhibited the growth of three ESCC cell lines, and this inhibition may have involved reductions in cyclin D1, Cdk4 and Cdk6.

Antidiabetic drug metformin inhibits esophageal adenocarcinoma cell proliferation in vitro and in vivo.

Esophageal carcinoma is the eighth most common cancer worldwide and the sixth leading cause of cancer-related deaths, with one of the worst prognoses of any form of cancer. Treatment with the anti-diabetic drug metformin has been associated with reduced cancer incidence in patients with type 2 diabetes. This study therefore evaluated the effects of metformin on the proliferation, in vitro and in vivo, of human esophageal adenocarcinoma cells, as well as the microRNAs associated with the antitumor effects of metformin. Metformin inhibited the proliferation of the esophageal adenocarcinoma cell lines OE19, OE33, SK-GT4 and OACM 5.1C, blocking the G0 to G1 transition in the cell cycle. This was accompanied by strong reductions in G1 cyclins, especially cyclin D1, cyclin-dependent kinase (Cdk)4, and Cdk6, and decreases in retinoblastoma protein phosphorylation. In addition, metformin reduced the phosphorylation of epidermal growth factor receptor and insulin-like growth factor and insulin-like growth factor-1 receptor, as well as angiogenesis-related proteins, such as vascular endothelial growth factor, tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2. Metformin also markedly altered microRNA expression. Treatment with metformin of athymic nude mice bearing xenograft tumors reduced tumor proliferation. These findings suggest that metformin may have clinical use in the treatment of esophageal adenocarcinoma.

Abundance of microRNA target motifs in the 3′‐UTRs of 20 527 human genes

A mechanism, selective avoidance, proposes that microRNA (miRNA) target sites are selectively depleted in the 3′‐UTRs of genes expressed at the same time and place as a miRNA. If this mechanism is ubiquitous, the target motif occurrences in the 3′‐UTRs would be decreased. To test this hypothesis, we examined miRNA target motif occurrences in the 3′‐ and 5′‐UTRs of 20 527 human protein‐coding genes. The results revealed that miRNA target motifs appeared more frequently than non‐target motifs and were enriched in the 3′‐UTRs. This enrichment was relatively reduced in a set of 2525 genes coexpressed with miR‐124a in the prefrontal cortex, but still remained at a high level, suggesting that miRNA target motifs are fostered by some other factors that surpass the influence of selective avoidance.