Regulation Of microRNA Expression Research Articles

Basic fibroblast growth factor promotes stem Leydig cell development and inhibits LH-stimulated androgen production by regulating microRNA expression

Leydig cells are the primary source of testosterone in the testes, and their steroidogenic function is strictly controlled by the hypothalamus–pituitary–gonad axis. Emerging evidence has indicated that fibroblast growth factors play a role in regulating stem Leydig cell development and steroidogenesis, but little is known about the regulatory mechanism. Using a seminiferous tubule culture system, we demonstrated that basic fibroblast growth factor (bFGF) can promote stem Leydig cell proliferation and commitment toward differentiation in testosterone-producing Leydig cells. However, these promoting effects decreased with an increase in the bFGF dose. Previous studies have reported that bFGF inhibits luteinizing hormone (LH)-stimulated androgen production by downregulating the mRNA expression of steroidogenic genes in immature Leydig cells. However, the expression levels of 677 microRNAs did not change significantly during the LH-mediated process of testosterone synthesis. Five microRNAs (miR-29a, -29c, -142-3p, -451 and -335) were identified, and their expression in immature Leydig cells was regulated simultaneously by bFGF and LH. These results suggested that the inhibition of LH-stimulated androgen production may be modulated by a change in bFGF-mediated microRNA expression, which further impacts the signaling pathway of testosterone biosynthesis and steroidogenic gene expression.

Abstract 147: δ-opioid Receptor Activation Increases Nrf2 Nuclear Translocation And Stabilizes Mitochondrial Membrane Potential Of Hypoxic Cells

Conditions that limit O2 supply for a prolonged period lead to cardiac injury. However, current strategies against hypoxic cardiac injury are limited in clinical settings. It is of utmost importance to find novel clues for protecting cells from hypoxic injury. Our work has recently shown that δ-opioid receptor (DOR) activation strongly regulates microRNA expression in the heart exposed to prolonged hypoxia, suggesting a role of DOR in cardiac adaptation to hypoxic stress. To further determine DOR's action on cell survival under hypoxia and the underlying mechanisms, we exposed HEK293t cells to hypoxia for a prolonged period and then investigated cellular viability and changes in mitochondrial membrane potential and the nuclear factor erythroid 2-related factor 2 (Nrf2), which are thought to be involved in the regulation of cell survival under hypoxia. Our data show that prolonged hypoxia (0.5% of O2 for 16-48 hours) caused serious cellular injury. DOR activation with UFP-512, a potent and specific DOR agonist, prevented the collapse of mitochondrial membrane potential and markedly attenuated cell injury in the hypoxic condition. The DOR-mediated protection was largely reversed by DOR antagonism with naltrindole. Furthermore, we observed that DOR activation increased Nrf2 translocation from cytoplasm to nucleus. The DOR-mediated cytoprotection was largely abolished by the "knock-down" of Nrf2. Our results suggest that DOR activation increases Nrf2 nuclear translocation and stabilizes mitochondrial membrane potential during the process of cellular protection against hypoxic stress. SC and TC are equal contributors. Supported by HD-034852, AT-004422, NSFC 81060096 and 81260204.

PDGF-regulated miRNA-138 inhibits the osteogenic differentiation of mesenchymal stem cells

Differentiation-specific microRNAs may play a critical role in MSC differentiation, and they can be altered by PDGF signaling. We propose that PDGF modulates MSC differentiation by regulating microRNA expression. Therefore, we investigated whether PDGF treatment could alter the expression profile of miRNAs in MSCs. Furthermore, we assessed the osteoblast phenotype of MSCs after inducing osteogenic differentiation. We found that PDGF treatment significantly inhibits the osteogenic differentiation of MSCs and that miR-138 gene transcription is controlled by PDGF signaling. Our results confirm that miR-138 inhibits the osteogenic differentiation of MSCs and suppresses the phosphorylation of FAK, ERK1/2, and Runx2. Furthermore, our study clearly demonstrates that downregulation of Runx2 by miR-138 is critical for the PDGF-mediated inhibition of osteogenic differentiation of MSCs. These findings indicate that inhibition of miR-138 function in MSCs, either by treatment with anti-miR-138 or by overexpression of the miR-138 target sequence (miRNA sponge), could represent a potential therapeutic strategy for the treatment of bone homeostasis disorders caused by activation of the PDGF pathway.

Regulation of microRNA expression in the lungs by exposure to air pollution & antigen (HUM2P.340)

Abstract Pulmonary Hypertension is characterized by pulmonary arterial remodeling and increased pressure in the pulmonary circulation. It is often associated with inflammation in the lungs and can lead to right heart failure. Our work shows that urban ambient pollution exacerbates the experimental pulmonary hypertension phenotype just like other types of inflammatory lung conditions. We aimed to identify microRNAs (miRNAs) that are differentially expressed in our mouse model. In addition, we examined plasma samples from individuals occupationally exposed to high levels of air pollution or cigarette smoke, and from controls. Our study is the first to show significantly de-regulated expression of three microRNA species (miR-135a, miR-21, miR-204) in the lungs of mice that were exposed to antigen and particulate matter and developed pulmonary hypertension. De-regulated levels of miR-21 and miR-204 have been reported in human pulmonary hypertension and in experimental pulmonary hypertension. MiR-135a is targeting STAT6 and upregulated expression has been reported in experimental asthma. Using human samples, our study showed that plasma levels of miR-21 and miR-135a, but not levels of miR-204, clustered individuals with high dose exposures and individuals with low dose environmental exposures. Current studies are aimed at identifying the cytokines that control these miRNAs’ expression. The long range goal is to identify miRNAs that indicate an at-risk state of the pulmonary vasculature.

Abstract W P201: Angiopoietin-1 Mimetic Peptide Treatment of Stroke Regulates MicroRNA Expression and Promotes Neuroprotection in Type One Diabetic Rats

Introduction: Angiopoietin-1 (Ang-1) mediates vascular remodeling. Diabetes decreases Ang1 and disrupts Ang1/Tie2 signaling, impairs vascular maturation and regulates immune response. MicroRNAs have been implicated in vascular diseases and inflammation. Vasculotide (VT) is an Ang1 mimetic peptide that promotes Tie2 activity. We hypothesize that treating type one diabetic rats (T1DM) with VT after stroke regulates miRNA expression decreases pro-inflammatory factors and thereby produces a neuroprotective effect. Methods: T1DM was induced in male Wistar rats with streptozotocin followed by 2h transient middle cerebral artery occlusion (MCAo). These rats were then treated with: 1) PBS control; 2) VT (3 ug/kg, ip injection) just prior to stroke and at 8h and 24h after MCAo. Functional tests were conducted 48h after MCAo. Afterwards, brain hemorrhage, BBB leakage, miRNA expression, RT-PCR, immunostaining and Western blot assays were done. Results: VT treatment did not decrease brain hemorrhage, but significantly decreased Evans Blue leakage (30.6±5.8 ng/mg vs. 17.9±2.0 ng/mg) lesion volume (35.1±3.5% vs 25.8±5.3%) and improved functional outcome after stroke when compared to control (p<0.05). To elucidate the mechanism, miRNA was measured. VT treatment significantly increased miR-451 (81.6 folds), miR-155 (2.4 fold), miR-98 (1.9 fold), and miR-126 (1.5 fold) expression, but decreased miR-200b (3.8 fold). Immunostaining showed that VT significantly decreased the number of apoptotic (45.5±3.7 vs 30.8±2.6) and cleaved-caspase-3 positive cells (45.2±3.5 vs 33.1±4.3), as well as monocyte chemotactic protein-1 (MCP1, 5.02±0.4% vs 1.8±0.2%) and tumor necrosis factor (TNF)-alpha (3.1±0.5% vs 1.5±0.2%). Western blot showed that VT significantly decreased receptor for advanced glycation end products (RAGE), MCP1 and TNFa (3-5 fold) in the ischemic border area compared to T1DM control. Conclusion: VT promotes neuroprotection after stroke in T1DM rats. Regulation of miRNA and neuroinflammatory factor expression and decreased BBB leakage may contribute to the VT induced neuroprotective effects observed after stroke in T1DM rats.

Molecular mechanism of hepatitis B virus-induced hepatocarcinogenesis.

Hepatitis B virus (HBV) infection is a global public health problem with approximately 2 billion people that have been exposed to the virus. HBV is a member of a family of small, enveloped DNA viruses called hepadnaviruses, and has a preferential tropism for hepatocytes of mammals and birds. Epidemiological studies have proved a strong correlation between chronic hepatitis B virus infection and the development of hepatocellular carcinoma (HCC). HCC is the fifth most common malignancy with about 700000 new cases each year, and more than 50% of them arise in HBV carriers. A large number of studies describe the way in which HBV can contribute to HCC development. Multiple mechanisms have been proposed, including the accumulation of genetic damage due to immune-mediated hepatic inflammation and the induction of oxidative stress. There is evidence of the direct effects of the viral proteins HBx and HBs on the cell biology. Integration of HBV-DNA into the human genome is considered an early event in the carcinogenic process and can induce, through insertional mutagenesis, the alteration of gene expression and chromosomal instability. HBV has also epigenetic effects through the modification of the genomic methylation status. Furthermore, the virus plays an important role in the regulation of microRNA expression. This review will summarize the many mechanisms involved in HBV-related liver carcinogenesis.

Regulation of microRNA expression in the neuronal stem cell niches during aging of the short-lived annual fish Nothobranchius furzeri

In the last decade, our group has intensively studied the annual fish Nothobranchius furzeri as a new experimental model in Biology specifically applied to aging research. We previously studied adult neuronal stem cells of N. furzeri in vivo and we demonstrated an age-dependent decay in adult neurogenesis. More recently we identified and quantified the expression of miRNAs in the brain of N. furzeri and we detected 165 conserved miRNAs and found that brain aging in this fish is associated with coherent up-regulation of well-known tumor suppressor miRNAs, as well as down-regulation of well-known onco miRNAs~– In the present work we characterized the expression of miR-15a, miR-20a, and microRNA cluster 17–92 in the principal neurogenic niches of the brain of young and old subjects of N. furzeri, by using in situ hybridization techniques, together with proliferating-cell nuclear antigen immuno-staining for a simultaneous visualization of the neuronal progenitors. We found that: (1) the expression of miR-15a is higher in the brain of old subjects and concentrates mainly in the principal neurogenic niches of telencephalon and optic tectum, (2) the expression of miR-20a is higher in the brain of young subjects, but more widespread to the areas surrounding the neurogenic niches, (3) finally, the expression of the microRNA cluster 17–92 is higher in the brain of young subjects, concentrated mainly in the principal neurogenic niches of telencephalon and cerebellum, and with reduced intensity in the optic tectum. Taken together, our data show that these microRNAs, originally identified in whole-brain analysis, are specifically regulated in the stem cell niche during aging.

Integrated analysis of microRNA and mRNA expression and association with HIF binding reveals the complexity of microRNA expression regulation under hypoxia.

BackgroundIn mammalians, HIF is a master regulator of hypoxia gene expression through direct binding to DNA, while its role in microRNA expression regulation, critical in the hypoxia response, is not elucidated genome wide. Our aim is to investigate in depth the regulation of microRNA expression by hypoxia in the breast cancer cell line MCF-7, establish the relationship between microRNA expression and HIF binding sites, pri-miRNA transcription and microRNA processing gene expression.MethodsMCF-7 cells were incubated at 1% Oxygen for 16, 32 and 48 h. SiRNA against HIF-1α and HIF-2α were performed as previously published. MicroRNA and mRNA expression were assessed using microRNA microarrays, small RNA sequencing, gene expression microarrays and Real time PCR. The Kraken pipeline was applied for microRNA-seq analysis along with Bioconductor packages. Microarray data was analysed using Limma (Bioconductor), ChIP-seq data were analysed using Gene Set Enrichment Analysis and multiple testing correction applied in all analyses.ResultsHypoxia time course microRNA sequencing data analysis identified 41 microRNAs significantly up- and 28 down-regulated, including hsa-miR-4521, hsa-miR-145-3p and hsa-miR-222-5p reported in conjunction with hypoxia for the first time. Integration of HIF-1α and HIF-2α ChIP-seq data with expression data showed overall association between binding sites and microRNA up-regulation, with hsa-miR-210-3p and microRNAs of miR-27a/23a/24-2 and miR-30b/30d clusters as predominant examples. Moreover the expression of hsa-miR-27a-3p and hsa-miR-24-3p was found positively associated to a hypoxia gene signature in breast cancer. Gene expression analysis showed no full coordination between pri-miRNA and microRNA expression, pointing towards additional levels of regulation. Several transcripts involved in microRNA processing were found regulated by hypoxia, of which DICER (down-regulated) and AGO4 (up-regulated) were HIF dependent. DICER expression was found inversely correlated to hypoxia in breast cancer.ConclusionsIntegrated analysis of microRNA, mRNA and ChIP-seq data in a model cell line supports the hypothesis that microRNA expression under hypoxia is regulated at transcriptional and post-transcriptional level, with the presence of HIF binding sites at microRNA genomic loci associated with up-regulation. The identification of hypoxia and HIF regulated microRNAs relevant for breast cancer is important for our understanding of disease development and design of therapeutic interventions.

Up-Regulation of miR-34a by Zinc Deficiency

Mild-to-moderate zinc deficiency is common throughout the world. The biochemical changes associated with zinc deficiency have been extensively studied. However, the study of the regulation of microRNAs expression by zinc has just begun. MicroRNAs play a significant role in the regulation of gene function by binding to complementary regions of specific target mRNAs. MicroRNAs whose expression is altered in response to low zinc may play a role in adaptive responses to low zinc. Herein, the effect of low zinc intake on the expression of miR-34a in intestine, liver and thymus was analyzed in mice. In addition, the effect of low and excess zinc on the expression of miR-34a was analyzed in human hepatoma cells HepG2. Mice fed a low zinc diet exhibited low zinc content in tibia and increased expression of zinc transporter Slc39a4 in intestine. Moreover, the expression of miR-34a was increased in intestine and thymus of zinc deficient mice. The expression of miR-34a was also increased in HepG2 cells grown in low zinc medium. However, the expression of this microRNA was not affected by excess zinc. The up-regulation of miR-34a associated with zinc deficiency could be part of an adaptive response to cues generated in cells under low zinc conditions such as oxidative stress and inflammation.

UVA and UVB Irradiation Differentially Regulate microRNA Expression in Human Primary Keratinocytes

MicroRNA (miRNA)-mediated regulation of the cellular transcriptome is an important epigenetic mechanism for fine-tuning regulatory pathways. These include processes related to skin cancer development, progression and metastasis. However, little is known about the role of microRNA as an intermediary in the carcinogenic processes following exposure to UV-radiation. We now show that UV irradiation of human primary keratinocytes modulates the expression of several cellular miRNAs. A common set of miRNAs was influenced by exposure to both UVA and UVB. However, each wavelength band also activated a distinct subset of miRNAs. Common sets of UVA- and UVB-regulated miRNAs harbor the regulatory elements GLYCA-nTRE, GATA-1-undefined-site-13 or Hox-2.3-undefined-site-2 in their promoters. In silico analysis indicates that the differentially expressed miRNAs responding to UV have potential functions in the cellular pathways of cell growth and proliferation. Interestingly, the expression of miR-23b, which is a differentiation marker of human keratinocytes, is remarkably up-regulated after UVA irradiation. Studying the interaction between miR-23b and its putative skin-relevant targets using a Luciferase reporter assay revealed that RRAS2 (related RAS viral oncogene homolog 2), which is strongly expressed in highly aggressive malignant skin cancer, to be a direct target of miR-23b. This study demonstrates for the first time a differential miRNA response to UVA and UVB in human primary keratinocytes. This suggests that selective regulation of signaling pathways occurs in response to different UV energies. This may shed new light on miRNA-regulated carcinogenic processes involved in UV-induced skin carcinogenesis.

Hormonal Regulation of MicroRNA Expression in Steroid Producing Cells of the Ovary, Testis and Adrenal Gland

BackgroundGiven the emerging roles of miRNAs as potential posttranscriptional/posttranslational regulators of the steroidogenic process in adrenocortical and gonadal cells, we sought to determine miRNA profiles in rat adrenals from animals treated with vehicle, ACTH, 17α-E2 or dexamethasone. Key observations were also confirmed using hormone (Bt2cAMP)-treated mouse Leydig tumor cells, MLTC-1, and primary rat ovarian granulosa cells.MethodologyRNA was extracted from rat adrenal glands and miRNA profiles were established using microarray and confirmed with qRT-PCR. The expression of some of the hormone-sensitive miRNAs was quantified in MLTC-1 and granulosa cells after stimulation with Bt2cAMP. Targets of hormonally altered miRNAs were explored by qRT-PCR and Western blotting in adrenals and granulosa cells.ResultsAdrenals from ACTH, 17α-E2 and dexamethasone treated rats exhibited miRNA profiles distinct from control animals. ACTH up-regulated the expression of miRNA-212, miRNA-182, miRNA-183, miRNA-132, and miRNA-96 and down-regulated the levels of miRNA-466b, miRNA-214, miRNA-503, and miRNA-27a. The levels of miR-212, miRNA-183, miRNA-182, miRNA-132, miRNA-370, miRNA-377, and miRNA-96 were up-regulated, whereas miR-125b, miRNA-200b, miR-122, miRNA-466b, miR-138, miRNA-214, miRNA-503 and miRNA27a were down-regulated in response to 17α-E2 treatment. Dexamethasone treatment decreased miRNA-200b, miR-122, miR-19a, miRNA-466b and miRNA27a levels, but increased miRNA-183 levels. Several adrenal miRNAs are subject to regulation by more than one hormone. Significant cAMP-induced changes in certain miRNAs were also noted in MLTC-1 and granulosa cells. Some of the hormone-induced miRNAs in steroidogenic cells were predicted to target proteins involved in lipid metabolism/steroidogenesis. We also obtained evidence that miR-132 and miRNA-214 inhibit the expression of SREBP-1c and LDLR, respectively.ConclusionOur results demonstrate that expression of a number of miRNAs in steroidogenic cells of the testis, ovary and adrenal glands is subject to hormonal regulation and that miRNAs and their regulation by specific hormones are likely to play a key role in posttranscriptional/posttranslational regulation of steroidogenesis.

NF90 in Posttranscriptional Gene Regulation and MicroRNA Biogenesis

Gene expression patterns are effectively regulated by turnover and translation regulatory (TTR) RNA-binding proteins (RBPs). The TTR-RBPs control gene expression at posttranscriptional levels, such as pre-mRNA splicing, mRNA cytoplasmic export, turnover, storage, and translation. Double-stranded RNA binding proteins (DSRBPs) are known to regulate many processes of cellular metabolism, including transcriptional control, translational control, mRNA processing and localization. Nuclear factor 90 (NF90), one of the DSRBPs, is abundantly expressed in vertebrate tissue and participates in many aspects of RNA metabolism. NF90 was originally purified as a component of a DNA binding complex which binds to the antigen recognition response element 2 in the interleukin 2 promoter. Recent studies have provided us with interesting insights into its possible physiological roles in RNA metabolism, including transcription, degradation, and translation. In addition, it was shown that NF90 regulates microRNA expression. In this review, we try to focus on the function of NF90 in posttranscriptional gene regulation and microRNA biogenesis.

Regulation of MicroRNA Expression by Rifampin in Human Hepatocytes

Rifampin causes drug interactions by altering hepatic drug metabolism. Because microRNAs (miRNAs) have been shown to regulate genes involved in drug metabolism, we determined the effect of rifampin on the expression of hepatic miRNAs. Primary human hepatocytes from seven subjects were treated with rifampin, and the expression of miRNA and cytochrome P450 (P450) mRNAs was measured by TaqMan assays and RNA-seq, respectively. Rifampin induced the expression of 10 clinically important and 13 additional P450 genes and repressed the expression of 9 other P450 genes (P < 0.05). Rifampin induced the expression of 33 miRNAs and repressed the expression of 35 miRNAs (P < 0.05). Several of these changes were highly negatively correlated with the rifampin-induced changes in the expression of their predicted target P450 mRNAs, supporting the possibility of miRNA-induced regulation of P450 mRNA expression. In addition, several other miRNA changes were positively correlated with the changes in P450 mRNA expression, suggesting similar regulatory mechanisms. Despite the interindividual variability in the rifampin effects on miRNA expression, principal components analysis clearly separated the rifampin-treated samples from the controls. In conclusion, rifampin treatment alters miRNA expression patterns in human hepatocytes, and some of the changes were correlated with the rifampin-induced changes in expression of the P450 mRNAs they are predicted to target.

C-Myc modulates microRNA processing via the transcriptional regulation of Drosha

The c-Myc oncogenic transcription factor is known to regulate microRNA (miRNA) expression at the transcriptional level. However, little is known about the function of c-Myc in miRNA processing. Here, we report that Drosha, one of the most important components of the miRNA processing machinery, is a c-Myc target gene. c-Myc transactivates drosha mRNA expression, thus upregulating the Drosha protein level. A chromatin immunoprecipitation (ChIP) experiment revealed that c-Myc binds directly to the E-box of the drosha promoter. Both in vitro and in vivo microRNA processing assays demonstrated that c-Myc promotes miRNA processing by upregulating the Drosha expression level. Overall, our study reveals a previously unrecognised function of c-Myc in miRNA processing and provides valuable insight into a new aspect of how c-Myc regulates microRNA expression.

Acetylation of Ets-1 Is the Key to Chromatin Remodeling for miR-192 Expression

By regulating gene expression, microRNAs (miRNAs) play pivotal roles in physiological and pathological processes. However, the regulation of miRNAs is elusive. miR-192 is a key regulator of renal fibrosis and hypertrophy in diabetic nephropathy. Natarajan et al. showed that the miR-192 gene contains an upstream region with Ets-1 and Smad3 binding sites. In control cells, all Ets-1 sites were occupied, resulting in a locked chromatin structure that kept miR-192 expression low. In response to transforming growth factor-β (TGF-β) stimulation, Smad3 and Akt were activated, and the latter further activated p300 to induce partial acetylation and dissociation of Ets-1 and the recruitment of Smad3 to the miR-192 gene, inducing transient miR-192 expression. During prolonged TGF-β treatment, p300 acetylated histone and Ets-1, resulting in complete dissociation of Ets-1 and the opening of the chromatin for sustained miR-192 expression. Thus, transcription factors and chromatin remodeling control microRNA gene expression in a dynamic, coordinated fashion.

Epigenetic regulation of microRNA expression in renal cell carcinoma

The underlying mechanisms of microRNA deregulation in cancer cells include epigenetic modifications, which play a crucial role in carcinogenesis. We demonstrate that numerous microRNAs are induced in renal cell carcinoma cell lines after treatment with inhibitors of the DNA-methyltransferase (5-aza-2′-deoxycytidine) and the histone-deacetylase (suberoylanilide hydroxamic acid). We provide evidence that enrichment of H3 and H3K18 acetylation at the miR-9 promoter is causative for re-expression, while DNA hypermethylation remains unchanged. Our experiments show that the treatment with the epigenetic drugs causes re-expression of silenced microRNAs with putative tumor suppressive function in ccRCC cell lines.

368 Regulation of MicroRNA Expression by Secretin During Cholestatic Liver Injury

368 Regulation of MicroRNA Expression by Secretin During Cholestatic Liver Injury

Abstract 3901: RBM3 drives tumor angiogenesis by modulating miR-210.

Abstract Background: Colorectal cancer is the second leading cause of cancer related deaths in the United States. While primary tumors can be resected out to inhibit progression, it is tumor metastasis that is deadly. A critical feature of metastatic spread of cells is tumor angiogenesis, wherein a network of blood vessels penetrates into the tumor and supplies nutrients and oxygen. Recently, we demonstrated that RNA binding protein RBM3 is a novel protooncogene that transforms normal cells to cancerous cells that can form xenografts in athymic nude mice. Here, we report that RBM3 induces angiogenesis through regulation of microRNA expression. Methods: Colon cancer cells HCT116, SW480 (Human) and CT26 (mouse), endothelial cells HUVEC, and the mouse fibroblast cells NIH3T3 were used in the study. RBM3 overexpressing cells were plated in soft agar and allowed to grow as spheroids, which were then injected into flanks of athymic nude mice. Total RNA and protein were isolated for Real Time RT-PCR and western blot analyses, respectively. Tumors were formalin fixed and used for immunohistochemistry. Microarray analyses were performed with total RNA from HCT116 cells overexpressing RBM3 to identify microRNAs that were differentially regulated. Results: Both HCT116 and SW480 cells demonstrated increased migration in scratch assay and in invasion assay when RBM3 is overexpressed. In addition, NIH3T3 and CT26 cells overexpressing RBM3 injected into the flanks of nude mice demonstrated increased metastasis to liver and lungs. There were also a significantly higher number of microvessels marked by CD31 endothelial cells within the primary tumor of the mice, within the tumor xenografts. In contrast, siRNA mediated knockdown of RBM3 expression in HCT116 tumor xenografts resulted in reduced tumor growth coupled with significant reduction in tumor microvasculature. RBM3 overexpression increased tube formation of HUVEC cells in matrigel tube formation assays. In contrast, RBM3 knockdown reduced tube formation. We next determined the effect of RBM3 expression on microRNA expression. Microarray studies showed that RBM3 overexpression in HCT116 cells resulted in increased levels of several microRNAs. miR210, a hypoxia-induced miRNA, is a critical angiogenesis regulator and endothelial cell survival factor whose overexpression stimulates angiogenesis and VEGF-induced cell migration. Microarray studies showed that miR210 was upregulated 3-fold in RBM3 overexpressing cells, which was validated by Real Time PCR analyses. miR210 was also upregulated in HUVEC cells overexpressing RBM3. Furthermore, the microRNA was reduced in HCT116 cells in culture and in tumor xenografts when RBM3 levels were downregulated by specific siRNA. Antagomir-mediated silencing of miR-210 significantly suppressed RBM3 mediated angiogenesis. Conclusion: RBM3 overexpression promotes tumor angiogenesis by upregulating miR-210 expression. Citation Format: Satish Ramalingam, dharmalingam Subramaniam, Shrikant Anant. RBM3 drives tumor angiogenesis by modulating miR-210. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3901. doi:10.1158/1538-7445.AM2013-3901

Abstract 5281: Insulin-mediated regulation of the miR132/212 cluster and miR 122/181a expression through PI3K, Akt, mTOR signaling in primary cultured rat hepatocytes.

Abstract Background and Objective: Several microRNAs were selected for characterization of their response to insulin signaling based on previous reports implicating their role in the regulation of hepatic metabolism, diabetes, hepatocellular carcinoma, and predictions of targeting CYP2E1. CYP2E1 catalyzes the metabolic activation of carcinogens, increases cellular oxidative stress, and is elevated in diabetes and at low insulin levels (0.1nM) in primary cultured hepatocytes. In addition, aberrant miR-122 expression was reported to be associated with hepatocellular carcinoma in rats, as well as in human tissue, and may be involved with hepatocarcinogenesis. In view of the critical role of the PI3K, Akt and mTOR signaling pathway in pathogenesis, mechanistic studies were conducted to examine the role of this pathway in regulation miR 132/212, 122 and 181a expression. Methods: Insulin effects on miRNA (miRs) levels were monitored using qPCR, and mechanistic studies employing inhibitors of PI3K, Akt, and mTOR were used to examine the role of this insulin signaling pathway on miR expression. The results were compared to miR let-7a, which is not implicated in the regulation of hepatic metabolism or diabetes and does not target CYP2E1 mRNA. Results: Insulin at concentrations of 0.1, 1.0, and 10 nM increased the expression of miRNA-132 and 212 ∼2- and 1.8-fold respectively, whereas expression of miRNAs 181a and 122 increased ∼1.6- and 1.4-fold, respectively. In contrast, insulin failed to significantly alter the expression of miRNA let-7a. Inhibition of PI3K with LY294002, Akt with A-443654 and mTOR with rapamycin resulted in significant suppression of the insulin-mediated elevation of miR-132, miR-212, and miR-122 levels monitored over the insulin concentrations examined. Inhibition of PI3K with LY294002, or Akt with A-443654 decreased the insulin-mediated increase in miR-181a levels by ∼20 to 50% relative to the respective insulin-concentration. Rapamycin inhibition of mTOR produced a marginal (&amp;lt;10%) decrease in miR-181a levels. Inhibition of PI3K or Akt did not show any significant effect on miR let-7a. In summary, seminal evidence is provided for insulin regulation of microRNA expression in primary cultured hepatocytes through PI3K, Akt and mTOR (TORC1). Citation Format: Upasana Shukla, Nithin Tumma, Theresa Gratsch, Alan Dombkowski, Raymond Novak. Insulin-mediated regulation of the miR132/212 cluster and miR 122/181a expression through PI3K, Akt, mTOR signaling in primary cultured rat hepatocytes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5281. doi:10.1158/1538-7445.AM2013-5281

MicroRNAs Profiling Reveals a Potential Link between the SDG8 Methyltransferase and Brassinosteroid-Regulated Gene Expression in Arabidopsis

Dong and Li, J Data Mining Genomics Proteomics 2013, 4:5http://dx.doi.org/10.4172/2153-0602.1000e110J Data Mining Genomics Proteomics Volume 4 • Issue 5 • 1000e110ISSN: 2153-0602 JDMGP, an open access journalMicroRNAs are a class of short non-coding RNAs (17-27 nucleotides) found in animals and plants. MicroRNAs play important roles in post-transcriptional regulation of gene expression by complementing the target mRNAs and causing translational repression or target mRNA degradation [1]. Studies have shown that thousands of human protein-coding genes are regulated by microRNAs [2], impacting many important biological processes, from development to physiology to stress response. In plants, microRNAs have been implicated in multiple essential biological processes, such as leaf morphogenesis and polarity, flower development, hormone signaling and metabolism, and stress responses [3]. MicroRNA genes are transcribed by RNA polymerases II, generating precursors that undergo a series of cleavage events to form mature microRNA [4]. MicroRNA biogenesis and its expression regulation are highly complicated [5]. Although the biological importance of microRNAs is well demonstrated in a wide range of cellular processes, how microRNA expression and abundance are regulated is not fully understood.Histone modifications play critical roles in regulation of gene expression in eukaryotes [6]. Acetylation of histones H3 and H4 is commonly associated with gene activation, whereas histone deacetylation generally leads to gene silencing. Trimethylation of histone H3 on lysine 4 (H3K4me3) is frequently associated with active transcription, while trimethylation of histone H3 on lysine 27 (H3K27me3) is often associated with gene repression. Trimethylation of histone H3 on lysine 36 (H3K36me3) is usually enriched in coding regions of actively transcribed genes [6], but H3K36me3 also correlates with gene silencing in facultative and constitutive heterochromatin[7].Recent studies in mammalian cells indicate that histone acetylation and methylation affect microRNA expression. Scott et al. [8] first showed that histone deacetylase inhibition results in alteration of microRNA levels in a breast cancer cell line. Following this study, several reports have shown that histone deacetylase inhibition alters microRNA expression in human carcinomas [9-11]. Overexpression of histone deacetylases in chronic lymphocytic leukemia results in silencing of miR-15a, miR-16, and miR-29b, while histone deacetylase inhibition can partially restore the expression of miR-15a, miR-16, and miR-29b[12]. These results collectively show that histone acetylation and deacetylation play important roles in regulation of microRNA expression in human cells. Stable RNAi-mediated suppression of the H3K4me3 demethylaseJARID1B in breast tumor cells caused increased expression of several members of the let-7 family of microRNAs, suggesting that H3K4me3 is required for up-regulation of the expression of these microRNAs [13]. Parallel sequencing analyses show that H3K27me3 is associated with repressed microRNA genes in mouse lymphocytes[14]. Taken together, these studies indicate that histone methylation can modulate microRNA expression.The SET domain group 8 (SDG8) protein is the primary methyltransferase for global histone H3K36 trimethylation in Arabidopsis [15,16]. SDG8 is involved in a number of developmental processes such shoot branching, ovule and anther development, and flowering time [16-18]. To explore the potential role of histone H3K36 trimethylation in the regulation of microRNA expression, we compared the miRNA expression profile of the knockout mutant of the SDG8 methyltransferase (sdg8) with the wild-type (WT) using microRNA microarray technology. MicroRNA microarray analysis was carried out by LC sciences (Houston, Texas, USA) on μParafloTM microfluidics chips containing 154 microRNA probes to