MicroRNAs (miRNAs) comprise species of short noncoding RNA that regulate gene expression post‐transcriptionally. Recent studies have demonstrated that epigenetic mechanisms, including DNA methylation and histone modification, not only regulate the expression of protein‐encoding genes, but also miRNAs, such as let‐7a, miR‐9, miR‐34a, miR‐124, miR‐137, miR‐148 and miR‐203. Conversely, another subset of miRNAs controls the expression of important epigenetic regulators, including DNA methyltransferases, histone deacetylases and polycomb group genes. This complicated network of feedback between miRNAs and epigenetic pathways appears to form an epigenetics–miRNA regulatory circuit, and to organize the whole gene expression profile. When this regulatory circuit is disrupted, normal physiological functions are interfered with, contributing to various disease processes. The present minireview details recent discoveries involving the epigenetics–miRNA regulatory circuit, suggesting possible biological insights into gene‐regulatory mechanisms that may underlie a variety of diseases.

Negative feedback is a crucial physiological regulatory mechanism, but no such regulator of angiogenesis has been established. Here we report a novel angiogenesis inhibitor that is induced in endothelial cells (ECs) by angiogenic factors and inhibits angiogenesis in an autocrine manner. We have performed cDNA microarray analysis to survey VEGF-inducible genes in human ECs. We characterized one such gene, KIAA1036, whose function had been uncharacterized. The recombinant protein inhibited migration, proliferation, and network formation by ECs as well as angiogenesis in vivo. This inhibitory effect was selective to ECs, as the protein did not affect the migration of smooth muscle cells or fibroblasts. Specific elimination of the expression of KIAA1036 in ECs restored their responsiveness to a higher concentration of VEGF. The expression of KIAA1036 was selective to ECs, and hypoxia or TNF-alpha abrogated its inducible expression. As this molecule is preferentially expressed in ECs, we designated it "vasohibin." Transfection of Lewis lung carcinoma cells with the vasohibin gene did not affect the proliferation of cancer cells in vitro, but did inhibit tumor growth and tumor angiogenesis in vivo. We propose vasohibin to be an endothelium-derived negative feedback regulator of angiogenesis.

Negative feedback is a crucial physiological regulatory mechanism, but no such regulator of angiogenesis has been established. Here we report a novel angiogenesis inhibitor that is induced in endothelial cells (ECs) by angiogenic factors and inhibits angiogenesis in an autocrine manner. We have performed cDNA microarray analysis to survey VEGF-inducible genes in human ECs. We characterized one such gene, KIAA1036, whose function had been uncharacterized. The recombinant protein inhibited migration, proliferation, and network formation by ECs as well as angiogenesis in vivo. This inhibitory effect was selective to ECs, as the protein did not affect the migration of smooth muscle cells or fibroblasts. Specific elimination of the expression of KIAA1036 in ECs restored their responsiveness to a higher concentration of VEGF. The expression of KIAA1036 was selective to ECs, and hypoxia or TNF-α abrogated its inducible expression. As this molecule is preferentially expressed in ECs, we designated it "vasohibin." Transfection of Lewis lung carcinoma cells with the vasohibin gene did not affect the proliferation of cancer cells in vitro, but did inhibit tumor growth and tumor angiogenesis in vivo. We propose vasohibin to be an endothelium-derived negative feedback regulator of angiogenesis.

The importance of microRNAs (miRNAs) in human malignancies has been well recognized. Here, we report that the expression of microRNA-210 (miR-210) is down-regulated in human esophageal squamous cell carcinoma and derived cell lines. Marked decreases in the level of miR-210 were observed especially in poorly differentiated carcinomas. We found that miR-210 inhibits cancer cell survival and proliferation by inducing cell death and cell cycle arrest in G 1 /G 0 and G 2 /M. Finally, we identified fibroblast growth factor receptor-like 1 (FGFRL1) as a target of miR-210 in esophageal squamous cell carcinoma and demonstrated that FGFRL1 accelerates cancer cell proliferation by preventing cell cycle arrest in G 1 /G 0 . Taken together, our findings show an important role for miR-210 as a tumor-suppressive microRNA with effects on cancer cell proliferation. MicroRNAs (miRNAs)2 are evolutionarily conserved small noncoding RNAs (20 -23 nucleotides) that bind to complementary sequences in the 3Ј-untranslated region (UTR) of target messenger RNAs (mRNAs) and regulate gene expression by the cleavage of target mRNAs and/or translational inhibition (1). Currently, Ͼ800 human miRNAs have been identified and registered in the miRNA database, miRBase (2). miRNAs play important roles in the differentiation of various cell types and in the initiation and progression of cancer, and it has been shown that the expression of some miRNAs is altered during cell differentiation and in malignancies (1, 3, 4).In a recent study, we identified microRNA-210 (miR-210) as one of the miRNAs that is markedly differentially expressed during the process of epithelial differentiation (3). It has been reported that miR-210 expression is down-regulated during epithelial-mesenchymal transition, the aberrant activation of which triggers cancer pathology (5). Carcinomas are derived from epithelial cells, and poor prognosis in patients with carcinoma is associated with the disruption of characteristics of differentiated epithelial cells, such as cell junctions and polarity (6 -8). Hence, given that the expression of miR-210 appears to be correlated well with epithelial differentiation, miR-210 might play a suppressive role in carcinomas. In support of this idea, allelic deletions at the miR-210 locus have been observed in 64% of cases of ovarian cancer (9), and ectopic expression of miR-210 represses tumor growth when human cancer cell lines are implanted into immunodeficient mice (10). However, the clinical roles of miR-210 in carcinomas and the mechanisms by which it represses tumor growth remain unknown.In this study, we investigated the functional role of miR-210 in the growth of carcinomas and the mechanism by which it acts using clinical samples as well as cell lines of esophageal squamous cell carcinoma (ESCC). ESCC is a highly aggressive malignancy with a 5-year survival rate of 10% worldwide. It has been used as a model to study the mechanisms of dysregulated epithelial differentiation and epithelial-mesenchymal transition in carcinomas (11, 12). E...

BackgroundCancer cells undergo massive alterations to their DNA methylation patterns that result in aberrant gene expression and malignant phenotypes. However, the mechanisms that underlie methylome changes are not well understood nor is the genomic distribution of DNA methylation changes well characterized.ResultsHere, we performed methylated DNA immunoprecipitation combined with high-throughput sequencing (MeDIP-seq) to obtain whole-genome DNA methylation profiles for eight human breast cancer cell (BCC) lines and for normal human mammary epithelial cells (HMEC). The MeDIP-seq analysis generated non-biased DNA methylation maps by covering almost the entire genome with sufficient depth and resolution. The most prominent feature of the BCC lines compared to HMEC was a massively reduced methylation level particularly in CpG-poor regions. While hypomethylation did not appear to be associated with particular genomic features, hypermethylation preferentially occurred at CpG-rich gene-related regions independently of the distance from transcription start sites. We also investigated methylome alterations during epithelial-to-mesenchymal transition (EMT) in MCF7 cells. EMT induction was associated with specific alterations to the methylation patterns of gene-related CpG-rich regions, although overall methylation levels were not significantly altered. Moreover, approximately 40% of the epithelial cell-specific methylation patterns in gene-related regions were altered to those typical of mesenchymal cells, suggesting a cell-type specific regulation of DNA methylation.ConclusionsThis study provides the most comprehensive analysis to date of the methylome of human mammary cell lines and has produced novel insights into the mechanisms of methylome alteration during tumorigenesis and the interdependence between DNA methylome alterations and morphological changes.

Objective-We recently isolated vasohibin, a novel vascular endothelial growth factor (VEGF)-inducible endotheliumderived angiogenesis inhibitor. Our aim is to find DNA sequences homologous to vasohibin and determine their expression profile. Methods and Results-By the search of DNA sequences in the database, we found one homologous gene and designated it vasohibin-2. Overall amino acid sequence homology between the prototype vasohibin (vasohibin-1) and vasohibin-2 was Ͼ50%. Vasohibin-2 exhibited antiangiogenic activity. Vasohibin-2 expression in cultured endothelial cells was low and not inducible by the stimulation that induced vasohibin-1. However, the immunohistochemical analysis revealed that vasohibin-1 and -2 were diffusely expressed in endothelial cells in embryonic organs during mid-gestation. After that time point, vasohibin-1 and -2 became faint, but persisted to a certain extent in arterial endothelial cells from late gestation to neonate. Expression of vasohibin-1 and -2 could be augmented in vivo by local transfection with the VEGF gene in the embryonic brain or by cutaneous wounding in adult mice. Conclusion-These results suggest that vasohibin-2, in combination with vasohibin-1, forms a novel family of angiogenesis inhibitors.

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ObjectiveTrastuzumab has been used for the treatment of HER2-positive breast cancer (BC). However, a subset of BC patients exhibited resistance to trastuzumab therapy. Thus, clarifying the molecular mechanism of trastuzumab treatment will be beneficial to improve the treatment of HER2-positive BC patients. In this study, we identified trastuzumab-responsive microRNAs that are involved in the therapeutic effects of trastuzumab.Methods and ResultsRNA samples were obtained from HER2-positive (SKBR3 and BT474) and HER2-negetive (MCF7 and MDA-MB-231) cells with and without trastuzumab treatment for 6 days. Next, we conducted a microRNA profiling analysis using these samples to screen those microRNAs that were up- or down-regulated only in HER2-positive cells. This analysis identified miR-26a and miR-30b as trastuzumab-inducible microRNAs. Transfecting miR-26a and miR-30b induced cell growth suppression in the BC cells by 40% and 32%, respectively. A cell cycle analysis showed that these microRNAs induced G1 arrest in HER2-positive BC cells as trastuzumab did. An Annexin-V assay revealed that miR-26a but not miR-30b induced apoptosis in HER2-positive BC cells. Using the prediction algorithms for microRNA targets, we identified cyclin E2 (CCNE2) as a target gene of miR-30b. A luciferase-based reporter assay demonstrated that miR-30b post-transcriptionally reduced 27% (p = 0.005) of the gene expression by interacting with two binding sites in the 3′-UTR of CCNE2.ConclusionIn BC cells, trastuzumab modulated the expression of a subset of microRNAs, including miR-26a and miR-30b. The upregulation of miR-30b by trastuzumab may play a biological role in trastuzumab-induced cell growth inhibition by targeting CCNE2.