Target Protein-coding Genes Research Articles

Enhancer Associated Long Non-coding RNA Transcription and Gene Regulation in Experimental Models of Rickettsial Infection.

Recent discovery that much of the mammalian genome does not encode protein-coding genes (PCGs) has brought widespread attention to long noncoding RNAs (lncRNAs) as a novel layer of biological regulation. Enhancer lnc (elnc) RNAs from the enhancer regions of the genome carry the capacity to regulate PCGs in cis or in trans. Spotted fever rickettsioses represent the consequence of host infection with Gram-negative, obligate intracellular bacteria in the Genus Rickettsia. Despite being implicated in the pathways of infection and inflammation, the roles of lncRNAs in host response to Rickettsia species have remained a mystery. We have profiled the expression of host lncRNAs during infection of susceptible mice with R. conorii as a model closely mimicking the pathogenesis of human spotted fever rickettsioses. RNA sequencing on the lungs of infected hosts yielded reads mapping to 74,964 non-coding RNAs, 206 and 277 of which were determined to be significantly up- and down-regulated, respectively, in comparison to uninfected controls. Following removal of short non-coding RNAs and ambiguous transcripts, remaining transcripts underwent in-depth analysis of mouse lung epigenetic signatures H3K4Me1 and H3K4Me3, active transcript markers (POLR2A, p300, CTCF), and DNaseI hypersensitivity sites to identify two potentially active and highly up-regulated elncRNAs NONMMUT013718 and NONMMUT024103. Using Hi-3C sequencing resource, we further determined that genomic loci of NONMMUT013718 and NONMMUT024103 might interact with and regulate the expression of nearby PCGs, namely Id2 (inhibitor of DNA binding 2) and Apol10b (apolipoprotein 10b), respectively. Heterologous reporter assays confirmed the activity of elncRNAs as the inducers of their predicted PCGs. In the lungs of infected mice, expression of both elncRNAs and their targets was significantly higher than mock-infected controls. Induced expression of NONMMUT013718/Id2 in murine macrophages and NONMMUT024103/Apol10b in endothelial cells was also clearly evident during R. conorii infection in vitro. Finally, shRNA mediated knock-down of NONMMUT013718 and NONMMUT024103 elncRNAs resulted in reduced expression of endogenous Id2 and Apl10b, demonstrating the regulatory roles of these elncRNAs on their target PCGs. Our results provide very first experimental evidence suggesting altered expression of pulmonary lncRNAs and elncRNA-mediated regulation of PCGs involved in immunity and during host interactions with pathogenic rickettsiae.

Cross-regulation of non-coding RNAs and their correlations with target protein-coding genes in CRC pathobiology

Illuminating the correlations between non-coding RNAs and protein-coding genes are of great interest to understand more about the molecular mechanisms that drive malignant transformation and understanding such correlations will enable development of more specific and efficient targeted therapeutics. Accordingly, in this comprehensive meta-analysis study, we tried to determine correlations between long non-coding RNA (lncRNA), microRNA (miRNA) and messenger RNA (mRNA) molecules that are involved in the pathogenesis of colorectal cancer (CRC). For the present study, current colorectal cancer studies published until 20 August 2017 and associated with the lncRNA-miRNA-mRNA interactions was included. The current literature search was done online in Pubmed, Embase and Web of Science databases. These databases have been screened with three keywords; “lncRNA”, “miRNA” and “colorectal cancer”. As a result, colorectal neoplasia differentially expressed (CRNDE) was determined to be consistently up-regulated in CRC tissues and inversely associated with miR-181a-5p. Also, CRNDE expression was significantly associated with lymph node metastasis (p = 0.032). Additionally, a significant association was determined between CRC and survival time in the OS analysis of FER1L4, TUSC7, UCC and lincRNA-ROR. More importantly, there was a negative correlation between lncRNA-miRNA expressions (p = 0.001). Particularly, CRNDE/miR-181a-5p, FER1L4/miR-106a-5p, TUSC7/miR-211, UCC/miR-143 and lincRNA-ROR/miR-145 was negatively correlated. In addition, there was a significant positive correlation between GAPLINC/CD44 and TUSC7/CDK6 in CRC tissues (p = 0.000). Overall analysis showed that lncRNAs and mRNAs, which are targets of the same miRNA, have positive interactions. In addition, miRNAs were shown to have negative correlations with target lncRNA/mRNA.

Systematic identification of long non-coding RNAs during pollen development and fertilization in Brassica rapa.

The importance of long non-coding RNAs (lncRNAs) in plant development has been established, but a systematic analysis of lncRNAs expressed during pollen development and fertilization has been elusive. We performed a time series of RNA-seq experiments at five developmental stages during pollen development and three different time points after pollination in Brassica rapa and identified 12051 putative lncRNAs. A comprehensive view of dynamic lncRNA expression networks underpinning pollen development and fertilization was provided. B.rapa lncRNAs share many common characteristics of lncRNAs: relatively short length, low expression but specific in narrow time windows, and low evolutionary conservation. Gene modules and key lncRNAs regulating reproductive development such as exine formation were uncovered. Forty-seven cis-acting lncRNAs and 451 trans-acting lncRNAs were revealed to be highly coexpressed with their target protein-coding genes. Of particular importance are the discoveries of 14 lncRNAs that were highly coexpressed with 10 function-known pollen-associated coding genes. Fifteen lncRNAs were predicted as endogenous target mimics for 13 miRNAs, and two lncRNAs were proved to be functional target mimics for miR160 after experimental verification and shown to function in pollen development. Our study provides the systematic identification of lncRNAs during pollen development and fertilization in B.rapa and forms the foundation for future genetic, genomic, and evolutionary studies.

Long non-coding RNA expression patterns in lung tissues of chronic cigarette smoke induced COPD mouse model

Long non-coding RNAs (lncRNAs) have critical regulatory roles in protein-coding gene expression. Aberrant expression profiles of lncRNAs have been observed in various human diseases. In this study, we investigated transcriptome profiles in lung tissues of chronic cigarette smoke (CS)-induced COPD mouse model. We found that 109 lncRNAs and 260 mRNAs were significantly differential expressed in lungs of chronic CS-induced COPD mouse model compared with control animals. GO and KEGG analyses indicated that differentially expressed lncRNAs associated protein-coding genes were mainly involved in protein processing of endoplasmic reticulum pathway, and taurine and hypotaurine metabolism pathway. The combination of high throughput data analysis and the results of qRT-PCR validation in lungs of chronic CS-induced COPD mouse model, 16HBE cells with CSE treatment and PBMC from patients with COPD revealed that NR_102714 and its associated protein-coding gene UCHL1 might be involved in the development of COPD both in mouse and human. In conclusion, our study demonstrated that aberrant expression profiles of lncRNAs and mRNAs existed in lungs of chronic CS-induced COPD mouse model. From animal models perspective, these results might provide further clues to investigate biological functions of lncRNAs and their potential target protein-coding genes in the pathogenesis of COPD.

Identification of Gossypium hirsutum long non-coding RNAs (lncRNAs) under salt stress

BackgroundLong non-coding RNAs (lncRNAs) represent a class of riboregulators that either directly act in long form or are processed into shorter microRNAs (miRNAs) and small interfering RNAs. Long noncoding RNAs (lncRNAs) are arbitrarily defined as RNA genes larger than 200 nt in length that have no apparent coding potential. lncRNAs have emerged as playing important roles in various biological regulatory processes and are expressed in a more tissue-specific manner than mRNA. Emerging evidence shows that lncRNAs participate in stress-responsive regulation.ResultsIn this study, in order to develop a comprehensive catalogue of lncRNAs in upland cotton under salt stress, we performed whole-transcriptome strand-specific RNA sequencing for three-leaf stage cotton seedlings treated with salt stress (S_NaCl) and controls (S_CK). In total we identified 1117 unique lncRNAs in this study and 44 differentially expressed RNAs were identified as potential non-coding RNAs. For the differentially expressed lncRNAs that were identified as intergenic lncRNAs (lincRNA), we analysed the gene ontology enrichment of cis targets and found that cis target protein-coding genes were mainly enriched in stress-related categories. Real-time quantitative PCR confirmed that all selected lincRNAs responsive to salt stress. We found lnc_388 was likely as regulator of Gh_A09G1182. And lnc_883 may participate in regulating tolerance to salt stress by modulating the expression of Gh_D03G0339 MS_channel. We then predicted the target mimics for miRNA in Gossypium. six miRNAs were identified, and the result of RT-qPCR with lncRNA and miRNA suggested that lnc_973 and lnc_253 may regulate the expression of ghr-miR399 and ghr-156e as a target mimic under salt stress.ConclusionsWe identified 44 lincRNAs that were differentially expressed under salt stress. These lincRNAs may target protein-coding genes via cis-acting regulation. We also discovered that specifically-expressed lincRNAs under salt stress may act as endogenous target mimics for conserved miRNAs. These findings extend the current view on lincRNAs as ubiquitous regulators under stress stress.

Genome-wide profiling of transcribed enhancers during macrophage activation

BackgroundMacrophages are sentinel cells essential for tissue homeostasis and host defence. Owing to their plasticity, macrophages acquire a range of functional phenotypes in response to microenvironmental stimuli, of which M(IFN-γ) and M(IL-4/IL-13) are well known for their opposing pro- and anti-inflammatory roles. Enhancers have emerged as regulatory DNA elements crucial for transcriptional activation of gene expression.ResultsUsing cap analysis of gene expression and epigenetic data, we identify on large-scale transcribed enhancers in bone marrow-derived mouse macrophages, their time kinetics, and target protein-coding genes. We observe an increase in target gene expression, concomitant with increasing numbers of associated enhancers, and find that genes associated with many enhancers show a shift towards stronger enrichment for macrophage-specific biological processes. We infer enhancers that drive transcriptional responses of genes upon M(IFN-γ) and M(IL-4/IL-13) macrophage activation and demonstrate stimuli specificity of regulatory associations. Finally, we show that enhancer regions are enriched for binding sites of inflammation-related transcription factors, suggesting a link between stimuli response and enhancer transcriptional control.ConclusionsOur study provides new insights into genome-wide enhancer-mediated transcriptional control of macrophage genes, including those implicated in macrophage activation, and offers a detailed genome-wide catalogue of transcribed enhancers in bone marrow-derived mouse macrophages.

Water-deficit stress-responsive microRNAs and their targets in four durum wheat genotypes.

MicroRNAs (miRNAs) guide regulation at the post-transcriptional level by inducing messenger RNA (mRNA) degradation or translational inhibition of their target protein-coding genes. Durum wheat miRNAs may contribute to the genotypic water-deficit stress response in different durum varieties. Further investigation of the interactive miRNA-target regulatory modules and experimental validation of their response to water stress will contribute to our understanding of the small RNA-mediated molecular networks underlying stress adaptation in durum wheat. In this study, a comprehensive genome-wide in silico analysis using the updated Triticum transcriptome assembly identified 2055 putative targets for 113 conserved durum miRNAs and 131 targets for four novel durum miRNAs that putatively contribute to genotypic stress tolerance. Predicted mRNA targets encode various transcription factors, binding proteins and functional enzymes, which play vital roles in multiple biological pathways such as hormone signalling and metabolic processes. Quantitative PCR profiling further characterised 43 targets and 5 miRNAs with stress-responsive and/or genotype-dependent differential expression in two stress-tolerant and two stress-sensitive durum genotypes subjected to pre-anthesis water-deficit stress. Furthermore, a 5' RLM-RACE approach validated nine mRNA targets cleaved by water-deficit stress-responsive miRNAs, which, to our knowledge, has not been previously reported in durum wheat. The present study provided experimental evidence of durum miRNAs and target genes in response to water-deficit stress in contrasting durum varieties, providing new insights into the regulatory roles of the miRNA-guided RNAi mechanism underlying stress adaptation in durum wheat.

Phage display and targeting peptides: surface functionalization of nanocarriers for delivery of small non-coding RNAs.

Small non-coding RNAs are known as a clinically relevant category of non-coding RNAs (ncRNAs) that have gained growing attention for their therapeutic values. miRNA and siRNA are of the most important and well-documented types of small ncRNAs. These regulatory RNA molecules are short–approximately 22 nucleotides long–with a similar mechanism of generation in which they are excised from longer double-stranded RNA precursor molecules. Both miRNA and siRNA target protein-coding genes via an antisense-based strategy and follow the same processing manner to reach gene-silencing effect (Chen et al., 2015). Functionally, small ncRNAs play a determining part in the modulation of cellular gene expression. The impressive role of these molecules as the controlling machinery of cells has proposed promising capacities for their potential translation into the clinic. In this context, tuning the regulatory components (such as ncRNAs) rather than the regulated components (protein-coding gens) has been suggested to be a more convenient and more effective approach to correct many cellular dysfunctions (Taft et al., 2010). This emerging concept symbolizes the dramatic potential of small ncRNAs, as a significant part of the regulatory apparatus of cells, to treat human diseases.

DNA Methylation of Tumor-Suppressor miRNA Genes in Chronic Lymphocytic Leukemia

DNA methylation is one of the most important epigenetic modifications of the genome involved in the regulation of numerous cellular processes through gene silencing without altering DNA sequences. miRNAs, a class of single-stranded noncoding RNAs of 19-25 nucleotides in length, function as post-transcriptional regulators of gene expression leading to mRNA cleavage or translational repression of their corresponding target protein-coding genes. Recently, dysregulation of tumor suppressor miRNAs mediated by promoter DNA hypermethylation is implicated in human cancers, including B-cell chronic lymphocytic leukemia (CLL). Moreover, it appears that methylated miRNA genes could be potential biomarkers for CLL diagnosis or therapy. This review will highlight the role of aberrant methylation of miRNA genes in the pathogenesis of CLL.

Methylation of miRNA genes and oncogenesis.

Interaction between microRNA (miRNA) and messenger RNA of target genes at the posttranscriptional level provides fine-tuned dynamic regulation of cell signaling pathways. Each miRNA can be involved in regulating hundreds of protein-coding genes, and, conversely, a number of different miRNAs usually target a structural gene. Epigenetic gene inactivation associated with methylation of promoter CpG-islands is common to both protein-coding genes and miRNA genes. Here, data on functions of miRNAs in development of tumor-cell phenotype are reviewed. Genomic organization of promoter CpG-islands of the miRNA genes located in inter- and intragenic areas is discussed. The literature and our own results on frequency of CpG-island methylation in miRNA genes from tumors are summarized, and data regarding a link between such modification and changed activity of miRNA genes and, consequently, protein-coding target genes are presented. Moreover, the impact of miRNA gene methylation on key oncogenetic processes as well as affected signaling pathways is discussed.

Multiple Targeting Routes of MicroRNA-22 Inform Cancer Drug Discovery

MicroRNAs (miRNAs) are a class of ~23-nt endogenous noncoding RNA transcripts that mediate the posttranscriptional repression of target protein-coding genes . Although the first evidence for the role of miRNAs in cancer development was presented in 2002 , the clinical trials of miRNA-based cancer drugs is just burgeoning recently ]. Certain miRNA has been shown to be capable of targeting multiple mRNAs and affecting extensive protein output . Particularly, miRNA-22 (miR-22) is one of the most studied multitasking miRNAs in tumor initiation and progression to date and attracts special attention from basic and clinical research scientists.

The Role of microRNAs in Mitochondria: Small Players Acting Wide.

MicroRNAs (miRNAs) are short, single-stranded, non-coding RNA molecules that act as post-transcriptional gene regulators. They can inhibit target protein-coding genes, through repressing messenger RNA (mRNA) translation or promoting their degradation. miRNAs were initially found to be originated from nuclear genome and exported to cytosol; where they exerted most of their actions. More recently, miRNAs were found to be present specifically in mitochondria; even originated there from mitochondrial DNA, regulating in a direct manner genes coding for mitochondrial proteins, and consequently mitochondrial function. Since miRNAs are recognized as major players in several biological processes, they are being considered as a key to better understand, explain, and probably prevent/cure not only the pathogenesis of multifactorial diseases but also mitochondrial dysfunction and associated diseases. Here we review some of the molecular mechanisms purported for miRNA actions in several biological processes, particularly the miRNAs acting in mitochondria or in mitochondria-related mechanisms.

Unbiased analysis of potential targets of breast cancer susceptibility loci by Capture Hi-C.

Genome-wide association studies have identified more than 70 common variants that are associated with breast cancer risk. Most of these variants map to non-protein-coding regions and several map to gene deserts, regions of several hundred kilobases lacking protein-coding genes. We hypothesized that gene deserts harbor long-range regulatory elements that can physically interact with target genes to influence their expression. To test this, we developed Capture Hi-C (CHi-C), which, by incorporating a sequence capture step into a Hi-C protocol, allows high-resolution analysis of targeted regions of the genome. We used CHi-C to investigate long-range interactions at three breast cancer gene deserts mapping to 2q35, 8q24.21, and 9q31.2. We identified interaction peaks between putative regulatory elements (“bait fragments”) within the captured regions and “targets” that included both protein-coding genes and long noncoding (lnc) RNAs over distances of 6.6 kb to 2.6 Mb. Target protein-coding genes were IGFBP5, KLF4, NSMCE2, and MYC; and target lncRNAs included DIRC3, PVT1, and CCDC26. For one gene desert, we were able to define two SNPs (rs12613955 and rs4442975) that were highly correlated with the published risk variant and that mapped within the bait end of an interaction peak. In vivo ChIP-qPCR data show that one of these, rs4442975, affects the binding of FOXA1 and implicate this SNP as a putative functional variant.

A germline mutation in the miR‑125a coding region reduces miR‑125a expression and is associated with human gastric cancer.

MicroRNAs (miRNAs) are small non-coding RNAs that inhibit the expression of target protein-coding genes, most often at the post-transcriptional level. miRNAs are often found to be misregulated in human cancer and they can act as potent oncogenes or tumor suppressor genes. In this study, we found that a germline mutation in the miR-125a coding region is associated with human gastric cancer. This mutation reduced the expression of mature miR-125a and alleviated its inhibitory effect on erythroblastic leukemia viral oncogene homolog 2 (ERBB2) gene expression and on gastric tumor cell proliferation. Thus, the data of this study suggested that this germline mutation in pri‑miR-125a likely contributes to the genetic predisposition to gastric cancer by reducing the production of miR-125a, thereby interfering with the expression of miR-125a target genes.

A Comprehensive Profile of ChIP-Seq-Based PU.1/Spi1 Target Genes in Microglia.

Microglia are resident mononuclear phagocytes that play a principal role in the maintenance of normal tissue homeostasis in the central nervous system (CNS). Microglia, rapidly activated in response to proinflammatory stimuli, are accumulated in brain lesions of neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease. The E26 transformation-specific (ETS) family transcription factor PU.1/Spi1 acts as a master regulator of myeloid and lymphoid development. PU.1-deficient mice show a complete loss of microglia, indicating that PU.1 plays a pivotal role in microgliogenesis. However, the comprehensive profile of PU.1/Spi1 target genes in microglia remains unknown. By analyzing a chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) dataset numbered SRP036026 with the Strand NGS program, we identified 5,264 Spi1 target protein-coding genes in BV2 mouse microglial cells. They included Spi1, Irf8, Runx1, Csf1r, Csf1, Il34, Aif1 (Iba1), Cx3cr1, Trem2, and Tyrobp. By motif analysis, we found that the PU-box consensus sequences were accumulated in the genomic regions surrounding ChIP-Seq peaks. By using pathway analysis tools of bioinformatics, we found that ChIP-Seq-based Spi1 target genes show a significant relationship with diverse pathways essential for normal function of monocytes/macrophages, such as endocytosis, Fc receptor-mediated phagocytosis, and lysosomal degradation. These results suggest that PU.1/Spi1 plays a crucial role in regulation of the genes relevant to specialized functions of microglia. Therefore, aberrant regulation of PU.1 target genes might contribute to the development of neurodegenerative diseases with accumulation of activated microglia.

The Intestinal Microbiota Interferes with the microRNA Response upon Oral Listeria Infection

ABSTRACTThe intestinal tract is the largest reservoir of microbes in the human body. The intestinal microbiota is thought to be able to modulate alterations of the gut induced by enteropathogens, thereby maintaining homeostasis. Listeria monocytogenes is the agent of listeriosis, an infection transmitted to humans upon ingestion of contaminated food. Crossing of the intestinal barrier is a critical step of the infection before dissemination into deeper organs. Here, we investigated the role of the intestinal microbiota in the regulation of host protein-coding genes and microRNA (miRNA or miR) expression during Listeria infection. We first established the intestinal miRNA signatures corresponding to the 10 most highly expressed miRNAs in the murine ileum of conventional and germfree mice, noninfected and infected with Listeria. Next, we identified 6 miRNAs whose expression decreased upon Listeria infection in conventional mice. Strikingly, five of these miRNA expression variations (in miR-143, miR-148a, miR-200b, miR-200c, and miR-378) were dependent on the presence of the microbiota. In addition, as is already known, protein-coding genes were highly affected by infection in both conventional and germfree mice. By crossing bioinformatically the predicted targets of the miRNAs to our whole-genome transcriptomic data, we revealed an miRNA-mRNA network that suggested miRNA-mediated global regulation during intestinal infection. Other recent studies have revealed an miRNA response to either bacterial pathogens or commensal bacteria. In contrast, our work provides an unprecedented insight into the impact of the intestinal microbiota on host transcriptional reprogramming during infection by a human pathogen.

Collaboration Between MiR-125b, MiR-100 and MiR-99a Induces Vincristine Resistance in Childhood TEL-AML1-Positive Acute Lymphoblastic Leukemia

Abstract 1475MicroRNAs (miRNAs) are involved in the pathobiology of leukemia. We previously observed that miR-125b, miR-99a and miR-100 were highly expressed in ex vivo vincristine (VCR) resistant leukemic cells of children with precursor B-cell acute lymphoblastic leukemia (ALL) (Schotte et al. Haematologica 2011). VCR is a microtubulin-interfering drug applied in the treatment of children with newly diagnosed ALL. Leukemic cells of TEL-AML1-positive precursor B-cell ALL were found to be resistant to this drug (Ramakers-van Woerden et al. Blood 2000). The expression levels of miR-125b, miR-99a and miR-100 were highly correlated in childhood precursor B-ALL cells. MiR-125b, as the most well-known miRNA among these three, was shown to contribute to drug resistance in different types of solid tumors. Down-regulation of miR-125b was also shown to sensitize the TEL-AML1-positive Reh cell line to doxorubicin and staurosporine (Gefen et al. Leukemia 2010). However, recent studies suggest that miRNAs co-operate and the combination of multiple distinct miRNAs co-regulates expression of associated target genes. The current study addressed whether miR-125b alone or in combination with miR-99a and/or miR-100 contributed to VCR resistance in childhood TEL-AML1-positive ALL cells.MiRNAs were over-expressed in TEL-AML1-positive Reh cells using lentiviral particles and/or miRNA precursors. Expression level of mature (and hence processed precursor) miRNAs was analyzed by qRT-PCR. The cell cycle distribution and the amount of leukemic cells in apoptosis were determined by flow cytometry of propidium iodide stained nuclei and Annexin V-propidium iodide stained cells, respectively. Leukemic cells were incubated with 9 ng/mL VCR and after 3 days of exposure the cellular response to VCR was measured by an MTT-based cell survival assay.The expression level of mature miR-125b, miR-99a and miR-100 was raised >100-fold upon lentiviral and precursor miRNA transduction compared to basal expression levels of TEL-AML1-positive Reh cells. Over-expression of miR-125b, miR-99a or miR-100 as single miRNAs did not significantly affect cellular survival after 3 days of exposure to VCR (20±5%, 29±17% and 29±17% viable cells, respectively) compared to the cells transduced with a scrambled miR-control (10±4% viable cells, p>0.05 each) when cell viability in absence of VCR was set to 100%. Over-expression of the combination of miR-100 and miR-99a only had a limited protective effect on cell viability (30±7% viable cells compared to 10±4% viable cells for the scrambled miR-control, p<0.05). In contrast, co-expression of miR-99a or miR-100 together with miR-125b or the combination of miR-99a and miR-100 together with miR-125b strongly and significantly induced resistance to VCR in TEL-AML- positive Reh cells; 91±4% of miR-99a/miR-125b-transduced, 93±5% of miR-100/miR-125b-transduced and 82±17% of miR-99a/miR-100/miR-125b-transduced cells remained alive upon VCR exposure compared to only 38±13% of miR-125b-transduced cells (p<0.05). The combination of these miRNAs did not change the cell cycle distribution or the amount of apoptotic cells in the absence of VCR. This suggests that these 3 miRNAs synergize in the development of resistance to VCR. Computational miRNA target prediction algorithms (TargetScan and EIMMo3) predicted 14 possible protein-coding target genes for the combination of miR-125b, miR-99a and miR-100. Gene expression profiling of ex vivo VCR-resistant (n=20) and VCR-sensitive (n=10) leukemic cells of TEL-AML1-positive ALL patients revealed that the expression levels of these 14 predicted target genes did not differ between resistant and sensitive cases (p>0.05). Ongoing studies currently explore which other protein-coding genes can be directly targeted by the miR125b, miR-99a and/or miR100 combination using functional gene expression studies.In conclusion, miR-125b, miR-99a or miR-100 as single factors were not effective to induce VCR resistance. In contrast, miR-125b in combination with miR-99a and/or miR-100 strongly increased VCR resistance in leukemic cells of TEL-AML1-positive precursor B-ALL. Discovery of directly regulated protein-coding target genes of these 3 miRNAs may point to ways to modulate resistance to VCR in children with ALL. Disclosures:No relevant conflicts of interest to declare.

Involvement of methylation of group of miRNA genes in regulation of expression of RAR-beta2 and NKIRAS1 target genes in lung cancer

To date, there are more than 2000 known human miRNAs, each of which may be involved in the regulation of hundreds of protein-coding target genes. In turn, the methylation of CpG islands affects the miRNA gene expression. Our aim was to evaluate the role of methylation in the regulation of miRNA gene expression and, consequently, in the regulation of the expression of target genes in primary lung tumors. Using a common collection of samples of non-small-cell lung cancer, we have performed a comprehensive study, including an analysis of the methylation status and level of expression of some miRNA genes and their potential target genes on chromosome 3, i.e., RAR-beta2 and NKIRAS1. The increased frequency of methylation in lung tumors compared to histologically normal tissue was revealed for miR-9-1 and miR-34b/c genes with significant statistics (P < 0.05 by Fisher’s exact test) and for miR-9-3 and miR-193a was marginally significant (P < 0.1). A significant correlation was revealed between the changes in methylation and level of expression of miR-9-1 gene (P ≈ 5 × 10−12 by Spearman) in lung tumors, which suggests the role of methylation in the regulation of expression of these miRNA genes. Furthermore, a statistically significant negative correlation (P ≈ 3 × 10−12 to 5 × 10−13 by Spearman) was found between changes in the levels of expression of miR-9-1 and miR-17 and RAR-beta2 target genes, as well as between the changes in the level of expression of miR-17 and NKIRAS1 that were not previously analyzed. The inverse relationship between the levels of expression of miRNA genes and their target genes is consistent with the known mechanism of the suppression of the expression of protein-coding genes under the action of miRNA. For the first time, significant correlations (P ≈ 3 × 10−10 to 4 × 10−13 by Spearman) were shown between changes in the methylation status of miRNA genes (miR-9-1, miR-9-3, miR-34b/c, miR-193a) and the level of expression of the RAR-beta2 target gene and changes in the methylation status of miR-34b/c, and miR-193a and the level of expression of the NKIRAS1 target gene in the primary lung tumors, which suggests the possibility of indirect effects of the methylation of miRNA genes on the level of expression of target genes.

Liver tumorigenicity promoted by microRNA-221 in a mouse transgenic model

MicroRNA-221 (miR-221) is one of the most frequently and consistently up-regulated microRNAs (miRNAs) in human cancer. It has been hypothesized that miR-221 may act as a tumor promoter. To demonstrate this, we developed a transgenic (TG) mouse model that exhibits an inappropriate overexpression of miR-221 in the liver. Immunoblotting and immunostaining confirmed a concomitant down-regulation of miR-221 target proteins. This TG model is characterized by the emergence of spontaneous nodular liver lesions in approximately 50% of male mice and by a strong acceleration of tumor development in 100% of mice treated with diethylnitrosamine. Similarly to human hepatocellular carcinoma, tumors are characterized by a further increase in miR-221 expression and a concomitant inhibition of its target protein-coding genes (i.e., cyclin-dependent kinase inhibitor [Cdkn]1b/p27, Cdkn1c/p57, and B-cell lymphoma 2-modifying factor). To validate the tumor-promoting effect of miR-221, we showed that in vivo delivery of anti-miR-221 oligonucleotides leads to a significant reduction of the number and size of tumor nodules. This study not only establishes that miR-221 can promote liver tumorigenicity, but it also establishes a valuable animal model to perform preclinical investigations for the use of anti-miRNA approaches aimed at liver cancer therapy.

Progranulin Regulator miR-107 Shows Cross-Species Homology with Plant Viruses (P05.054)

Objective: Our hypothesis is that plant viruses augment or suppress endogenous human miRNAs and consequently the target protein-coding genes, thus contributing to the pathogenesis of human disease or helping to preserve health. In the current study, our objective was to uncover plant viruses that are homologous or complementary to human miRNAs. Background Progranulin (PRGN) is a neuroprotective protein that is expressed in both neurons and microglia in the CNS and is associated with frontotemporal lobar degeneration (FTLD) and Alzheimer9s disease (AD). Human microRNAs (miRNA) are short non-coding RNA molecules of 20-23 nucleotides that regulate gene expression. A specific micro RNA, miR-107, is down-regulated in AD and has recently been shown to target PRGN expression. Design/Methods: We performed a Basic Local Alignment Search Tool (BLAST) analysis to find nucleotide sequences similarities in two databases: 1) the moRBase Sequence Database, a repository of published sequences and associated annotation, and 2) the Descriptions of Plant Viruses (DPVweb) genome database. Sequences that showed a statistically significant homology or complementarity of >12 nucleotides (equal or larger than 50% of the average miR length) were selected for functional and gene ontology categorization. Results: miR-107 showed sequence similarity to Grapevine berry virus A (15 base homology), Grapevine berry inner necrosis virus (15 base homology), Soybean dwarf virus (14 base complementarity) and Cymbidium ringspot virus (13 base complementarity). Conclusions: This is the first bioinformatics demonstration of molecular mimicry between plant viruses and human miRNAs. These data support our hypothesis that certain viral nucleotides show sequence homology with a miRNA that has been linked to FTLD and AD. Exposure to plant viruses may influence miR-107 mediated post-transcriptional regulation of PRGN. Finally, the discovery of plant viruses that mimic miR-107 might provide a novel strategy for targeted treatment of FTLD, AD and other neurodegenerative conditions. Disclosure: Dr. Vaishnav has nothing to disclose. Dr. Rebolledo-Mendez has nothing to disclose. Dr. Raha-Chowdhury has nothing to disclose. Dr. Nandi has nothing to disclose. Dr. Roberts has nothing to disclose. Dr. Palmer has nothing to disclose. Dr. Cooper has nothing to disclose. Dr. Friedland has nothing to disclose.