microRNA-210 Research Articles

Abstract 17288: GTP Cyclohydrolase I Rescues microRNA-34a Impairment of Endothelial Progenitor Cell Angiogenesis in Aging

Introduction: Endothelial progenitor cells (EPCs) are critically regulated by eNOS for their angiogenic functions. eNOS and its essential cofactor tetrahydrobiopterin (BH4) are reduced during aging, and aged EPCs exhibit decreased proliferation, migration and survival. We recently reported that microRNA 34a (miR-34a) induces EPC senescence and inhibits its angiogenesis by targeting silent information regulator 1 (SIRT1). However, whether miR-34a regulation of EPC angiogenesis involves eNOS and BH4 in aging is unknown. We hypothesized that miR-34a promotes impairment of EPC angiogenesis in aging, which could be rescued by GTP cyclohydrolase I (GTPCH I, the rate-limiting enzyme for BH4 biosynthesis). Methods: Bone marrow-derived EPCs from young (8-10 weeks) C57BL/6 (wild type, WT), GTPCH I transgenic (Tg-GCH, endothelial-specific GTPCH I overexpression), hph-1 (BH4-deficient), aged (18-20 months) C57BL/6 (WT) and eNOS/GCH double-transgenic (Tg-eNOS/GCH) mice (all males) were used. Results: Aged WT mice showed significantly impaired recovery of hindlimb ischemia, compared with young WT mice (0.40±0.13 vs. 0.68±0.12, n=3, p<0.05). Consistently, EPCs from aged WT mice showed impaired Matrigel angiogenesis (0.46±0.03 vs. 1.00±0.04, n=4, p<0.01). Reactive oxygen species (ROS) level was significantly elevated in EPCs from aged WT mice (~1.6 folds vs. young WT mice, n=3, p<0.05), accompanied by reduced GTPCH I expression (0.63±0.07 vs. 1.00±0.08, n=3, p<0.05). Suppression of miR-34a in EPCs from aged WT mice rescued their impaired angiogenesis, while overexpression of miR-34a in EPCs from young WT mice impaired their angiogenesis in vitro. Moreover, miR-34a expression was decreased in EPCs from aged Tg-eNOS/GCH double-transgenic mice vs. aged WT mice (0.83±0.07 vs. 1.91±0.07, n=3, p<0.01), with a concomitant augmentation of SIRT1 level (1.14±0.02 vs. 0.54±0.06, n=3, p<0.01). Finally, miR-34a expression was down-regulated in EPCs from Tg-GCH mice with an increase in SIRT1 level, and was up-regulated in EPCs from BH4-deficient hph-1 mice with a decrease in SIRT1 level vs. WT mice. Conclusion: These findings demonstrate, for the first time, that miR-34a promotes impairment of EPC angiogenesis in aging, which could be rescued by GTPCH I/BH4.

Modulation of MicroRNA 34a in Early Colon Carcinogenesis: Potential Mechanism for Gender Related Initiation-Progression Discordance

Modulation of MicroRNA 34a in Early Colon Carcinogenesis: Potential Mechanism for Gender Related Initiation-Progression Discordance

Systematic Proteome Analysis Identifies Transcription Factor YY1 as a Direct Target of miR-34a

MicroRNA 34a (miR-34a) is a potential tumor suppressor gene and has been identified as a miRNA component of the p53 network. To better understand the biological pathways involved in miR-34a action, a parallel global protein and mRNA expression profiling on miR-34a treated neuroblastoma cells (IMR32) was performed using isotope-coded affinity tags (ICAT) and Affymetrix U133plus2 microarray, respectively. Global profiling showed that miR-34a causes much smaller mRNA expression changes compared to changes at the protein level. A total of 1495 proteins represented by two or more peptides were identified from the quantitative ICAT analysis, of which 143 and 192 proteins are significantly up- or down-regulated by miR-34a, respectively. Pathway analysis of these differentially expressed proteins showed the enrichment of apoptosis and cell death processes in up-regulated proteins but DNA replication and cell cycle processes in the down-regulated proteins. Ribosomal proteins are the most significant set down-regulated by miR-34a. Additionally, biological network analysis to identify direct interactions among the differentially expressed proteins demonstrated that the expression of the ubiquitous transcription factor YY1, as well as its downstream proteins, is significantly reduced by miR-34a. We further demonstrated that miR-34a directly targets YY1 through a miR-34a-binding site within the 3' UTR of YY1 using a luciferase reporter system. YY1 is a negative regulator of p53, and it plays an essential role in cancer biology. Therefore, YY1 is another important direct target of miR-34a which closely regulates TP53 activities.

The Silencing of MicroRNA 148a Production by DNA Hypermethylation Is an Early Event in Pancreatic Carcinogenesis

The poor prognosis of pancreatic ductal adenocarcinoma (PDAC) is accounted for by the absence of early diagnostic markers and effective treatments. MicroRNAs inhibit the translation of their target mRNAs. The production of microRNAs is strongly altered in cancers, but the causes of these alterations are only partially known. DNA hypermethylation is a major cause of gene inactivation in cancer. Our aims were to identify microRNAs whose gene expression is inactivated by hypermethylation in PDAC and to determine whether this hypermethylation-mediated repression is an early event during pancreatic carcinogenesis. We also sought to investigate whether these differentially methylated regions can serve as a diagnostic marker for PDAC. MicroRNA production was measured by microarray hybridization and reverse-transcription quantitative PCR. The level of DNA methylation was measured by bisulfite mapping and semiquantitative methylation-specific PCR. We identified 29 microRNAs encoded by genes whose expression is potentially inactivated by DNA hypermethylation. We focused our study on microRNA 148a (miR-148a) and found its production to be repressed, not only in PDAC samples but also in preneoplastic pancreatic intraepithelial neoplasia (PanIN) lesions. More importantly, we found that hypermethylation of the DNA region encoding miR-148a is responsible for its repression, which occurs in PanIN preneoplastic lesions. Finally, we show that the hypermethylated DNA region encoding miR-148a can serve as an ancillary marker for the differential diagnosis of PDAC and chronic pancreatitis (CP). We show that the hypermethylation of the DNA region encoding miR-148a is responsible for its repression in PDAC precursor lesions and can be a useful tool for the differential diagnosis of PDAC and CP.

Abstract 2101: GBM: miR-34a acts as a tumor suppressor and prognosis maker as well as modulating EGFR

Abstract The whole genomic gene copy number was measured using single nucleotide polymorphism DNA microarray (SNP-Chip). Chromosome 1p36.23 was frequently deleted (19 of 55 clinical samples, 35 %) and EGFR was often amplified (35 of 55, clinical samples 66 %) in glioblastoma multiform (GBM). microRNA 34a (miR-34a) localizes in this minimum common deleted 1p36.23 region. We hypothesized that miR-34a may act as a tumor suppressor in the brain. Interestingly, we found that the mean survival time was significantly shorten (p < 0.05) in the patients whose GBM showed both EGFR amplification and miR-34a deletion compared to those with either EGFR amplification, miR-34a deletion or normal levels of EGFR and miR-34a. Expression of miR-34a was significantly lower in GBM samples compared to normal brain tissue as examined using real-time RT PCR. Forced expression of miR-34a in GBM cell lines decreased their cell growth both in liquid culture and in immunodeficient mice; and the latter was associated with decreased angiogenesis. These cells also had decreased ability to migrate as detected by Boyden chamber and had profoundly low levels of cell cycle proteins (Cyclin -A1, -D1, -D3 and -B1, CDK2, as well as, E2F1, −3, and −4) and increased expression of cyclin kinase inhibitor (CKI) proteins, p21 and p27. Furthermore, the protein expression of EGFR was decreased in the cells with overexpression of miR-34a. Taken together, GBMs often acquire a miR-34a deletion and have low expression of this microRNA. The miR-34a probably normally acts as a tumor suppressor by inhibiting growth of GBM cells in vitro and in vivo associated with moderating the expression of cell cycle proteins and EGFR. Individuals whose GBM have both deletion of miR-34a and amplification of EGFR have a particularly poor prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2101.

Mir-210 and target mRNA expression in the kidney after ischemic-reperfusion injury (IRI)

Objective To investigate the effect of mir-210 and target mRNA Ephrin-A3 expres-sion in the kidney after ischemic-reperfusion injury (IRI) in mouse. Methods Ten KunMing mice were randomly divided into 2 groups, namely ischemia-reperfusion 4 h group (IR group) and sham operation group(sham group). For mice in IR group,bilateral renal pedicles were clamped for 40 min. For those in sham group, the kidneys were simply separated and exposed for 40 min. After the operation for 4 h, mice were sacrificed and kidney samples were examined for Ephrin-A3 gene by RT-PCR, Ephrin-A3 protein by-Immunochemistry,miR-210 by quantitative Real-Time RT-PCR. Results Ephrin-A3 expressed in tubular epithelia. In IR group,renal Ephrin-A3 gene and miR-210 expression were higher than in Sham group, Ephrin-A3 protein expression decreased compared with Sham group. Conclusion The renal ischemia/reperfusion injury might put an influence on the expression of mir-210 and Ephrin-A3 mRNA. The repair of renal ischemia/reperfusion injury might be associated with the alternation of miR-210 expression. Key words: Ischemia-reperfusion renal; MicroRNA-210; Ephrin-A3

The Role of MICRORNA 34-A During Apoptosis-Mediated Mouse Neural Stem Cell Differentiation

The Role of MICRORNA 34-A During Apoptosis-Mediated Mouse Neural Stem Cell Differentiation

MiR-34a repression of SIRT1 regulates apoptosis

MicroRNA 34a (miR-34a) is a tumor suppressor gene, but how it regulates cell proliferation is not completely understood. We now show that the microRNA miR-34a regulates silent information regulator 1 (SIRT1) expression. MiR-34a inhibits SIRT1 expression through a miR-34a-binding site within the 3' UTR of SIRT1. MiR-34 inhibition of SIRT1 leads to an increase in acetylated p53 and expression of p21 and PUMA, transcriptional targets of p53 that regulate the cell cycle and apoptosis, respectively. Furthermore, miR-34 suppression of SIRT1 ultimately leads to apoptosis in WT human colon cancer cells but not in human colon cancer cells lacking p53. Finally, miR-34a itself is a transcriptional target of p53, suggesting a positive feedback loop between p53 and miR-34a. Thus, miR-34a functions as a tumor suppressor, in part, through a SIRT1-p53 pathway.

Osteogenic Differentiation of Human Adipose Tissue-Derived Stem Cells Is Modulated by the miR-26a Targeting of the SMAD1 Transcription Factor

The molecular mechanisms that regulate hADSC differentiation toward osteogenic precursors and subsequent bone-forming osteoblasts is unknown. Using osteoblast precursors obtained from subcutaneous human adipose tissue, we observed that microRNA-26a modulated late osteoblasts differentiation by targeting the SMAD1 transcription factor. Elucidation of the molecular mechanisms guiding human adipose tissue-derived stem cells (hADSCs) differentiation is of extreme importance for improving the treatment of bone-related diseases such as osteoporosis. The aim of this study was to identify microRNA as a regulator of the osteogenic differentiation of hADSCs. Osteoblast differentiation of hADSCs was induced by treatment with dexamethasone, ascorbic acid, and beta-glycerol phosphate. The expression of osteoblastic phenotype was evaluated after the induction by simultaneous monitoring of alkaline phosphatase activity, the expression of genes involved in osteoblastic differentiation by real-time RT-PCR, and mineralization at the same time. MicroRNA expression was determined by Northern blot, and transfection of both antisense miR-RNA and sensor plasmids was done to validate the inhibitory role of microRNA during hADSC osteogenesis. Western blot was used to determine the expression levels of the SMAD1 protein. qRT-PCR analysis was used to compare the expression patterns of osteoblastic markers in transfected cells. We analyzed the role of microRNA 26a (miR-26a) during differentiation of hADSCs. Northern blot analysis of miR-26a during hADSC differentiation showed increased expression, whereas expression of the SMAD1 protein was complementary to that of miR-26a. Because the highest expression of miR-26a and the lowest expression of SMAD1 protein were reached at hADSC terminal differentiation, we carried out our study during the late stages of hADSC differentiation. The inhibition of miR-26a, by 2'-O-methyl-antisense RNA, increased protein levels of its predicted target, SMAD1 transcription factor, in treated osteoblasts, upregulating bone marker genes and thus enhancing osteoblast differentiation. Our data suggest a role for miR-26a in the differentiation induced by treatment with dexamethasone, ascorbic acid, and beta-glycerol phosphate of hADSCs toward the osteogenic lineage by targeting its predicted target, the SMAD1 protein. This study contributes to a better knowledge of molecular mechanisms governing hADSC differentiation by proposing a microRNA-based control of late differentiation.