Stranded RNA Molecules Research Articles

Comprehensive secondary structure elucidation of four genera of the family Pospiviroidae.

Viroids are small, circular, single stranded RNA molecules that infect plants. Since they are non-coding, their structures play a critical role in their life cycles. To date, little effort has been spend on elucidating viroid structures in solution due to both the experimental difficulties and the time-consuming nature of the methodologies implicated. Recently, the technique of high-throughput selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) was adapted for the probing of the members of family Avsunviroidae, all of whom replicate in the chloroplast and demonstrate ribozyme activity. In the present work, twelve viroid species belonging to four different genera of the family Pospiviroidae, whose members are characterized by the presence of a central conserved region (CCR) and who replicate in nucleus of the host, were probed. Given that the structures of five distinct viroid species from the family Pospiviroidae have been previously reported, an overview of the different structural characteristics for all genera and the beginning of a manual classification of the different viroids based on their structural features are presented here.

Swimming training restores the cardiac microRNA‐1, and ‐214 levels and cardiac contractility in rat after myocardial infarction (LB43)

Myocardial infarction (MI) is accompanied by cardiac hypertrophy and fibrosis related to ventricular compliance reduction. MicroRNAs (miRNAs) are noncoding single stranded RNA molecules that regulate gene expression. The miRNA‐1, which target NCX and ‐214, which target Serca2a are involved in cardiac contractily. This study assessed the effects of swimming training (ST) on the cardiac miRNAs‐1 and ‐214 levels and ventricular function after MI. Male Wistar rats (n=6/group) were randomized into four groups: Control (CO), Training (TR), Control Infarcted (CI) Training Infarcted (TI). ST consisted of 60 min/days/5 days/week/10 week with 3% body overload began four weeks after the MI. Echocardiography was performed before and after the ST. MiRNAs analysis was performed by real‐time PCR in the Remote Myocardium (RM). The VO2max increased 38%in the TR compared with CO group and increased 14% in the TI compared with CI group. TI group decreased 15% the E/A ratio compared with CI group, showing improved ventricular compliance. Also, ST restored the cardiac shortening fraction in the TI group compared with CO group. MiRNA‐1 and ‐214 expression, respectively, were decreased (52%) and increased (54%) in the CI compared to the CO . However, ST increased (49%) and decreased (29%), respectively, the MiRNA‐1 and ‐214 expression in the TI compared to the CO . In this way, NCX and Serca2a were respectively decreased (36%) and increased (48%) in the TI compared to the CI. These results show that ST restored cardiac miRNA‐1 and ‐214 expression, which can be associate to the improved ventricular function, suggesting a mechanism for its potential therapeutic application in heart diseases.Grant Funding Source: Foundation for Research Support of the State of São Paulo ‐ FAPESP

Significance of microRNA targeted estrogen receptor in male fertility.

Estrogen receptor-alpha (ERα) mediates estrogen action in regulation of different levels of the hypothalamic-pituitary-testis axis. It has a key role in spermatogenesis. Estrogen receptor alpha knock-out (ER koα) male mice were infertile and severe impairment in spermatogenesis and seminiferous tubules was observed. Recently, it has been reported that microRNA (miRNA) mir-100 and let-7b were predicted to target ERα gene. MiRNA are small, endogenous, single stranded RNA molecules that regulate gene expression and have been implicated in various disease states. It has been proved that some miRNAs expression is tissue- and disease-specific, giving potential for identifying miRNAs as a diagnostic tool. In this study, the change in the expression levels of mir-100, let-7b and ERα expression levels were evaluated in oligospermic infertile patients (n=43) compared to control fertile subjects (n=43). After washing and separating sperms, total RNA was isolated and then cDNA was synthesized. The expression levels of mir-100 and let-7b and ERα were evaluated by real time PCR. Mir-100, let-7b levels were significantly higher than those in control group (P=0.008 and P=0.009, respectively). We have found that, ERα level was significantly decreased in comparison with normal group (P< 0.0001). Changes in mir-100, let-7b and ERα expression levels in oligospermic patients may be associated with the susceptibility and progression of infertility. The results of this study indicate that miRNA can have a key role in spermatogenesis and might have a diagnostic and prognostic value in men infertility.

Double-Strand RNA Binding Protein Profiling

The double-strand RNA-binding proteins (DRBP), which are featured by the existence of evolutionarily conserved dsRNA-binding domain (DRBD), have been reported to function in a variety of significant cellular activities, involving RNA processing, RNA cleavage, RNA interference and anti-viral immunity pathways.However, it still remains unclear about the molecular mechanism and dynamic properties underlying the interaction between DRBP and dsRNA. Recently single-molecule experiments have shown an ATP-independent diffusion/sliding activity of purified TRBP as well as its two homologs, PKR activator and R3D1-L, on double strand RNA molecules (Koh et al., 2013), indicating a potential generalizability of diffusion/sliding activity in dsRNA-binding proteins family. To test this hypothesis and to further characterize DRBPs in terms of substrate specificity, binding affinity, features of motion upon binding and multiple DRBD collaboration or redundancy, we examined in vivo expressed proteins with one or more DRBDs at single-molecule level.In this research, DRBP genes have been cloned from Human Open Reading Frame Library and overexpressed in human A549 cells. Then we immobilized the protein products on quartz slide surface by Single Molecule Pull Down (Jain et al. 2011) and investigated their dynamic property upon biding various dsRNA substrates using Protein Induced Fluorescence Enhancement (PIFE) (Hwang et al, 2011). PIFE data from one of our candidates, TRBP, showed the intensity fluctuation of Cy3, the fluorescent label of dsRNA substrates, indicating that TRBP is sliding back and forth along dsRNA strand upon binding to it (Fig. 1). This result is highly consistent with the experimental observation in vitro (Koh et al., 2013).1. Koh H. R et al. (2013) Proc. Natl. Acad. Sci USA 110:151-15.2. Jain A et al. (2012) Nat Protoc 7:445-452.3. Hwang H et al. (2011) Proc Natl Acad Sci USA 108: 7414-7418.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Potential Urinary miRNA Biomarker Candidates for the Accurate Detection of Prostate Cancer among Benign Prostatic Hyperplasia Patients

MicroRNAs (miRNAs) are a class of short (~22nt), single stranded RNA molecules that function as post-transcriptional regulators of gene expression. MiRNAs can regulate a variety of important biological pathways, including: cellular proliferation, differentiation and apoptosis. Profiling of miRNA expression patterns was shown to be more useful than the equivalent mRNA profiles for characterizing poorly differentiated tumours. As such, miRNA expression “signatures” are expected to offer serious potential for diagnosing and prognosing cancers of any provenance. The aim of this study was to investigate the potential of using deregulation of urinary miRNAs in order to detect Prostate Cancer (PCa) among Benign Prostatic Hyperplasia (BPH). To identify the miRNA signatures specific for PCa, miRNA expression profiling of 8 PCa patients, 12 BPH patients and 10 healthy males was carried out using whole genome expression profiling. Differential expression of two individual miRNAs between healthy males and BPH patients was detected and found to possibly target genes related to PCa development and progression. The sensitivity and specificity of miR-1825 for detecting PCa among BPH individuals was found to be 60% and 69%, respectively. Whereas, the sensitivity and specificity of miR-484 were 80% and 19%, respectively. Additionally, the sensitivity and specificity for miR-1825/484 in tandem were 45% and 75%, respectively. The proposed PCa miRNA signatures may therefore be of great value for the accurate diagnosis of PCa and BPH. This exploratory study has identified several possible targets that merit further investigation towards the development and validation of diagnostically useful, non-invasive, urine-based tests that might not only help diagnose PCa but also possibly help differentiate it from BPH.

Abstract LB-246: MicroRNA profiling can classify acute leukemias of ambiguous lineage as either acute myeloid leukemia or acute lymphoid leukemia.

Abstract Classification of acute leukemia (AL) is based on commitment of the leukemic cells to either the myeloid or the lymphoid lineage. However, a small percentage of AL cases display features of both hematopoietic lineages and lacks immunophenotypical lineage commitment. These leukemias of ambiguous lineage represent a heterogeneous category of AL that cannot be classified as either myeloid AL (AML) or lymphoid AL (ALL). The lack of a clear classification of acute leukemias of ambiguous lineage as either AML or ALL is a hurdle in treatment choice for these patients. MicroRNAs are small single stranded RNA molecules which regulate gene expression by promoting degradation of mRNAs or repressing their translation. MicroRNA expression profiles have shown to be able to accurately discriminate AL of the lymphoid lineage from those of the myeloid lineage. Here, we compared the microRNA expression profiles of acute leukemias of ambiguous lineage with those of B-ALL, T-ALL and AML. MicroRNA expression profiles of nine patients with leukemia of ambiguous lineage and eleven patients with AML, B-ALL or T-ALL were analyzed. Unsupervised clustering analysis of the AML, B-ALL and T-ALL samples resulted in a clear separation between cases of the myeloid and lymphoid lineages. The top differentially expressed microRNAs between AML and ALL were miR-199b, miR-27a/b, miR-223, miR-23a, miR-221 and miR-150. Subsequently, we compared the general miRNA expression profiles of AL of ambiguous lineage with those of the AML, B-ALL and T-ALL cases thereby showing that these leukemias of ambiguous lineage do not segregate as a separate entity. Moreover, unsupervised clustering of all the AL samples using the top 10 percent of most variable microRNAs resulted in clustering of leukemias of ambiguous lineage with either AML, B-ALL or T-ALL. Furthermor, qRT-PCR expression analysis of the five most discriminative microRNAs between ALL and AML was able to define AL of ambiguous lineage as either AML or ALL. Thus, our results indicate the presence of a lymphoid or myeloid lineage specific microRNA expression profile in AL cases of ambiguous lineage, despite their mixed immunophenotype. At diagnosis, the classification of AL of ambiguous lineage by microRNA expression analysis might be of additional value in therapeutic decision making. Citation Format: Dave C. de Leeuw, Willemijn van den Ancker, Fedor Denkers, Rene X. Menezes, Theresia M. Westers, Gert J. Ossenkoppele, Arjan A. van de Loosdrecht, Linda Smit. MicroRNA profiling can classify acute leukemias of ambiguous lineage as either acute myeloid leukemia or acute lymphoid leukemia. [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 LB-246. doi:10.1158/1538-7445.AM2013-LB-246

Abstract 4164: Resveratrol-induced apoptosis in breast cancer cells is mediated by tumor suppressor microRNA.

Abstract Background: Breast cancer is the second most common type of cancer in women, and the fifth most common cause of cancer related deaths in the world. Several drugs are being used for treatment, but it is seen that after prolonged use tumor cells develop resistance to these drugs rendering them ineffective. Identifying alternative and effective treatment options is crucial to improve the life expectancy of breast cancer patients. Resveratrol, a dietary agent found in grapes is being considered as a potential drug candidate due to its pro-apoptotic properties against cancer cells. Simultaneously, microRNAs (miRNAs) which are small non-coding single stranded RNA molecules are gaining ground as novel cancer therapeutics due to their capability to alter cell functions including apoptosis. Objective: The overall objective of this study is to elucidate that resveratrol-induced apoptosis in breast cancer cells occurs via tumor-suppressor miRNA. Experimental setup: Breast cancer cell lines MCF-7 (Estrogen Receptor positive) and MDA-MB-231 (Estrogen Receptor negative) were treated with different doses of resveratrol (0, 50, 100, 200 μM). Total RNA was isolated and miRNA analysis was performed using a breast cancer specific miRNA array to screen for miRNA known or predicted to have altered expression during breast cancer initiation or progression. A real-time PCR based on the SYBR Green technology was performed on these samples to study the expression profiles of 84 miRNAs using this method. Results: Analysis of cells after resveratrol treatment clearly showed an apoptotic effect on the cells in a dose dependent manner. Cell viability assay showed that the 50 μM dose had a protective effect with cells being more viable as compared to the control cells, but at consecutively higher doses resveratrol had a very distinct apoptotic effect. MiRNA data analysis revealed several miRNAs to be regulated in breast cancer cells. Hsa-miR-613 was found to be upregulated at both doses whereas hsa-miR-199b-5p was upregulated at 50 μM and downregulated at 200 μM in MCF-7. Similarly, for MDA-MB-231 cells hsa-miR -129-5p, hsa-miR-199b-3p and hsa-miR-199a-5p were upregulated at both doses whereas hsa-miR-214-3p was down regulated at 50 μM and upregulated at 200 μM. Interestingly, several members of the let-7 family were altered in both cell types. Several miRNAs associated with drug-resistance and ER dependence were also identified in both the cell types. Conclusion: Resveratrol induces apoptosis in breast cancer cells via the action of several miRNA. Therefore, treatment with resveratrol in conjunction with miRNAs that are altered by resveratrol has the potential to be a good therapeutic model to induce tumor suppression and ultimately destroy the tumors. Citation Format: Tanvi Muni, Anand Iyer, Neelam Azad. Resveratrol-induced apoptosis in breast cancer cells is mediated by tumor suppressor microRNA. [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 4164. doi:10.1158/1538-7445.AM2013-4164

Microrna Profiling Classifies Acute Leukemias of Ambiguous Lineage As Either Acute Myeloid Leukemia or Acute Lymphoid Leukemia

Abstract 1443 IntroductionClassification of acute leukemia (AL) is based on commitment of the leukemic cells to either the myeloid or the lymphoid lineage. However, a small percentage of AL cases lacks immunophenotypical lineage commitment or displays features of both hematopoietic lineages. These leukemias of ambiguous lineage represent a heterogeneous category of AL that cannot be classified as either myeloid AL (AML) or lymphoid AL (ALL). This presents a major hurdle for choice of treatment, because it is unsettled whether these patients benefit from AML or ALL treatment regimens or a mixture of both. Better diagnostic tools to define the lineage of origin for these AL would be instrumental to direct therapy decision making.MicroRNAs are small single stranded RNA molecules which regulate gene expression by promoting degradation of mRNAs or repressing their translation. MicroRNA expression profiles have been shown to accurately discriminate AL of the lymphoid lineage from AL of the myeloid lineage. Here, we investigated microRNA expression profiles of leukemias of ambiguous lineage to classify them as either AML or ALL. MethodsMicroRNA expression profiles of nine patients with leukemia of ambiguous lineage and eleven patients with AML, B-ALL or T-ALL were analyzed using microarray analysis. MicroRNAs differentially expressed between the myeloid and lymphoid lineage were selected using Linear Model for Microarray Analysis (LIMMA) and used for unsupervised clustering of the ambiguous lineage leukemias with the AML, B-ALL and T-ALL samples. The top five most discriminating microRNAs were selected for qRT-PCR validation in eight additional ambiguous lineage cases, five AML or ALL cases and two control cell lines. ResultsUnsupervised clustering analysis of the AML, B-ALL and T-ALL samples resulted in a clear separation between the myeloid and lymphoid lineages. Top differentially expressed microRNAs were miR-199b, miR-27a/b, miR-223, miR-23a, miR-221 and miR-150. When comparing leukemias of ambiguous lineage with AML, B-ALL and T-ALL using LIMMA, no clear differences were found, indicating that leukemias of ambiguous lineage are not a separate entity. Moreover, unsupervised clustering of all AL samples using the top 10 percent of most variable microRNAs resulted in clustering of leukemias of ambiguous lineage with either AML, B-ALL or T-ALL and showed comparable expression profiles. Expression analysis by qRT-PCR of the five most discriminative microRNAs on the additional samples, including leukemias of ambiguous lineage, was able to accurately assign these leukemias to the lineage of origin. ConclusionMicroRNA expression profiling of leukemias of ambiguous lineage indicated the presence of a myeloid or lymphoid lineage-specific genotype despite an indistinct immunophenotype. Analyzing microRNA expression at diagnosis to classify acute leukemias of ambiguous lineage as either AML or ALL might be instrumental to therapeutic decision making. Disclosures:No relevant conflicts of interest to declare.

Inhibition of 5′-UTR RNA Conformational Switching in HIV-1 Using Antisense PNAs

BackgroundThe genome of retroviruses, including HIV-1, is packaged as two homologous (+) strand RNA molecules, noncovalently associated close to their 5′-end in a region called dimer linkage structure (DLS). Retroviral HIV-1 genomic RNAs dimerize through complex interactions between dimerization initiation sites (DIS) within the (5′-UTR). Dimer formation is prevented by so calledLong Distance Interaction (LDI) conformation, whereas Branched Multiple Hairpin (BMH) conformation leads to spontaneous dimerization.Methods and ResultsWe evaluated the role of SL1 (DIS), PolyA Hairpin signal and a long distance U5-AUG interaction by in-vitro dimerization, conformer assay and coupled dimerization and template-switching assays using antisense PNAs. Our data suggests evidence that PNAs targeted against SL1 produced severe inhibitory effect on dimerization and template-switching processes while PNAs targeted against U5 region do not show significant effect on dimerization and template switching, while PNAs targeted against AUG region showed strong inhibition of dimerization and template switching processes.ConclusionsOur results demonstrate that PNA can be used successfully as an antisense to inhibit dimerization and template switching process in HIV -1 and both of the processes are closely linked to each other. Different PNA oligomers have ability of switching between two thermodynamically stable forms. PNA targeted against DIS and SL1 switch, LDI conformer to more dimerization friendly BMH form. PNAs targeted against PolyA haipin configuration did not show a significant change in dimerization and template switching process. The PNA oligomer directed against the AUG strand of U5-AUG duplex structure also showed a significant reduction in RNA dimerization as well as template- switching efficiency.The antisense PNA oligomers can be used to regulate the shift in the LDI/BMH equilibrium.

Genetic and epigenetic variants influencing the development of nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is common worldwide. The importance of genetic and epigenetic changes in etiology and pathogenesis of NAFLD has been increasingly recognized. However, the exact mechanism is largely unknown. A large number of single nucleotide polymorphisms (SNPs) related to NAFLD has been documented by candidate gene studies (CGSs). Among these genes, peroxisome proliferatoractivated receptor-γ, adiponectin, leptin and tumor necrosis factor-α were frequently reported. Since the introduction of genome-wide association studies (GWASs), there have been significant advances in our understanding of genomic variations of NAFLD. Patatin-like phospholipase domain containing family member A3 (PNPLA3, SNP rs738409, encoding I148M), also termed adiponutrin, has caught most attention. The evidence that PNPLA3 is associated with increased hepatic fat levels and hepatic inflammation has been validated by a series of studies. Epigenetic modification refers to phenotypic changes caused by an adaptive mechanism unrelated to alteration of primary DNA sequences. Epigenetic regulation mainly includes microRNAs (miRs), DNA methylation, histone modifications and ubiquitination, among which miRs are studied most extensively. miRs are small natural single stranded RNA molecules regulating mRNA degradation or translation inhibition, subsequently altering protein expression of target genes. The miR-122, a highly abundant miR accounting for nearly 70% of all miRs in the liver, is significantly under-expressed in NAFLD subjects. Inhibition of miR-122 with an antisense oligonucleotide results in decreased mRNA expression of lipogenic genes and improvement of liver steatosis. The investigation into epigenetic involvement in NAFLD pathogenesis is just at the beginning and needs to be refined. This review summarizes the roles of genetics and epigenetics in the development of NAFLD. The progress made in this field may provide novel diagnostic biomarkers and therapeutic targets for NAFLD management.

RNA Deep Sequencing Reveals Differential MicroRNA Expression during Development of Sea Urchin and Sea Star

microRNAs (miRNAs) are small (20–23 nt), non-coding single stranded RNA molecules that act as post-transcriptional regulators of mRNA gene expression. They have been implicated in regulation of developmental processes in diverse organisms. The echinoderms, Strongylocentrotus purpuratus (sea urchin) and Patiria miniata (sea star) are excellent model organisms for studying development with well-characterized transcriptional networks. However, to date, nothing is known about the role of miRNAs during development in these organisms, except that the genes that are involved in the miRNA biogenesis pathway are expressed during their developmental stages. In this paper, we used Illumina Genome Analyzer (Illumina, Inc.) to sequence small RNA libraries in mixed stage population of embryos from one to three days after fertilization of sea urchin and sea star (total of 22,670,000 reads). Analysis of these data revealed the miRNA populations in these two species. We found that 47 and 38 known miRNAs are expressed in sea urchin and sea star, respectively, during early development (32 in common). We also found 13 potentially novel miRNAs in the sea urchin embryonic library. miRNA expression is generally conserved between the two species during development, but 7 miRNAs are highly expressed in only one species. We expect that our two datasets will be a valuable resource for everyone working in the field of developmental biology and the regulatory networks that affect it. The computational pipeline to analyze Illumina reads is available at http://www.benoslab.pitt.edu/services.html.

P3-03-05: Identification of miRNAs and Their Associated Target Genes Involved in Endocrine Resistance in Breast Cancer.

Abstract Background: Despite the widespread use of the selective estrogen receptor modulator tamoxifen or strategies of estrogen deprivation in the treatment of estrogen receptor-positive breast cancer, tumor resistance to endocrine therapy remains a serious clinical problem. MiRNAs are a class of endogenous, single stranded RNA molecules that play a pivotal role in the regulation of gene expression. Altered expression levels of these molecules have been implicated in a number of malignancies, including breast cancer. The aim of this study was to identify those miRNAs that may be associated with endocrine resistance and to further investigate their role in development of this resistance. Methods: MCF7L breast cancer cells were cultured in regular parental medium (Par) or in phenol-red free, 10% charcoal-stripped FBS medium treated by estrogen deprivation (ED, &amp;gt;6months), tamoxifen (Tam, 10−7M, &amp;gt;6months), or estrogen (E2, 10−9M,&amp;gt;1month). Cells treated by long-term Tam or ED developed resistance (TamR or EDR) and resumed cell growth, which was confirmed by growth curve assay. Parallel short-term treatment (10 days) with estrogen deprivation or tamoxifen to represent the endocrine-sensitive phase, and short-term E2 treated cells as a control was also performed. A whole-transcriptome miRNA microarray was performed by High Throughput Genomics (Tucson, AZ), using cell lysates from Par, short-term/endocrine sensitive, E2 treated cells, TamR and EDR. The expression levels of several miRNAs were validated by real-time quantitative PCR (Q-PCR). MiRNAs dysregulated in TamR cells were integrated with predicted gene targets showing anti-correlated expression patterns across human breast tumors in The Cancer Genome Atlas (TCGA) datasets. Results: The miRNA microarray identified 23 miRNAs that are upregulated in TamR cells (&amp;gt;1.7 fold change, p&amp;lt;0.005) compared to parental cells grown in the presence of E2, and 37 miRNAs that were downregulated (&amp;gt;50%, p&amp;lt;0.005). In addition, 6 miRNAs were observed to be upregulated in EDR and 3 downregulated in EDR compared with controls. Validation of four of these upregulated miRNAs within the TamR group, miR221, miR-222, miR-301b and miR-181c, was performed using Q-PCR. Previously identified genes associated with TamR were scanned for potential binding sites of these deregulated miRNAs using miRanda and TargetScan and many potential miRNA targeted genes are downregulated in TamR. Both miR-221 and miR-181c are predicted to target estrogen receptor gene (ESR1), and downregulation of this target gene was confirmed at the mRNA level by Q-PCR. A strong negative correlation was identified between ESR1 and both miR-181c and miR-221 expression levels in breast tumor samples, according to TCGA data. Work is ongoing to identify the role of miR-181c and other miRNAs in tamoxifen resistance, using a loss-of-function approach. Parallel miRNA profiling of multiple endocrine models is underway to identify commonly deregulated miRNAs associated with endocrine resistance. Conclusions: This study has identified those miRNAs associated with endocrine resistance in breast cancer. These miRNAs may provide potential predictive markers of resistance to endocrine therapy and modulation of these may lead to increased therapeutic sensitivity. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-03-05.

Blockade of B7-H1 enhances dendritic cell-mediated T cell response and antiviral immunity in HBV transgenic mice

Dendritic cells (DCs) have been identified as the most effective antigen-presenting cell (APC), much attention has been directed toward the use of DCs in vaccine strategies for the treatment of cancer or chronic virus infection. B7-H1 (PD-L1) is a ligand for programmed cell death-1 (PD-1) and does not bind to other CD28 family members, it is expressed on resting and upregulated on activated B, T, myeloid, and dendritic cells (DCs). The overwhelming number of studies supports the role of B7-H1 as a negative regulator of T cell responses. RNA interference (RNAi) is a mechanism for sequence-specific posttranscriptional inhibition of gene expression via double stranded RNA molecules and it has recently been applied to mammalian cells with the use of small interfering RNAs (siRNAs). In the study, transfection of DCs with siRNA specific for B7-H1 gene resulted in the blockade of the expression of B7-H1 on DCs. The allostimulatory activity of DCs could be enhanced by silencing of B7-H1 on DCs. Blockade of B7-H1 on DCs inhibited the production of IFN-γ and IL-10 but not IL-2 and IL-4 in mixed lymphocyte reaction (MLR). HBV specific peptide-pulsed DCs could break tolerance and trigger specific CTL responses, the level of HBsAg and HBV DNA in sera of HBV transgenic mice decreased, whereas blockade of B7-H1 on DCs augmented the effects. These data strongly support the concept that blockade of B7-H1 can enhance DC-mediated antiviral immune responses.

MicroRNAs: A Novel Therapeutic Target for Schizophrenia

Schizophrenia is one of the most disabling psychiatric conditions. Current treatments target monoamine receptors but this approach does not address the full complexity of the disorder. Here we explore the possibility of developing new anti-psychotics by targeting microRNAs (miRNAs), single stranded RNA molecules, 21-23 nucleotides in length that are not translated into proteins and regulate gene expression. The present review reveals that research involving schizophrenia and miRNA is very recent (the earliest report from 2007) and miRNAs add a significant layer of complexity to the pathophysiology of the disorder. However, miRNAs offer an exciting potential not only to understand the underlying mechanisms of schizophrenia, but also for the future development of antipsychotics, as the human miRNA system provides a rich and diverse opportunity for pharmacological targeting. However, technology is still developing in order to produce effective strategies to modulate specific and localized changes in miRNA, particularly in relation to the central nervous system and schizophrenia.

Abstract 3016: The value of microRNAs in the detection and staging of colorectal cancer

Abstract Introduction: MicroRNAs (miRNAs) are a large class of single strand RNA molecules consistent of 18-25 nucleotides, involved in post transcriptional gene silencing. miRNAs show tissue-specific expression and play an important role in carcinogenesis. The aim of the present study was: A. to identify colorectal cancer (CRC)-specific miRNAs. B. To study the potential value of CRC-specific miRNAs in CRC detection and staging. Methods: This study was approved by the Institutional Review Board (IRB). RNA was extracted from paired samples of normal and tumor tissue of CRC patients (n=10). miRNA expression was tested using a commercially available array. Differential expression between normal and tumor tissues was identified in 30 human miRNAs and in additional 16 miRNAs identified from database mining. Differential expression was validated by quantitative (real-time) PCR in a larger cohort of samples (n=20). RNA was extracted from sentinel lymph nodes (SLN) of CRC patients. A calibration plot was generated using a CRC cell line (HT29) mixed with peripheral blood lymphocytes (PBL) of healthy donors in a range of concentrations. Results: After validation, only 8 miRNAs were found to be overexpressed in CRC. The detection threshold for all 8 miRNAs measured by the calibration plot was 1:1000 (one HT-29 cell in 1000 PBMCs). The expression of the 8 miRNAs was then tested in SLNs (n=86). Fourteen lymph nodes (16.3%) were positive by standard pathological examination (H&amp;E staining) and 25/86 (29.1%) were positive by miRNA expression analysis (p&amp;lt;0.01). miRNA expression was detected in all sera from CRC patients (n=3) and in none of the sera obtained from healthy individuals (n=5, p&amp;lt;0.0001). Conclusions: A panel of 8 miRNAs was found to be overexpressed in CRC patients. Occult residual disease was indentified in lymph nodes and serum of CRC patients using this miRNA panel. 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 3016.

Hantavirus Nucleocapsid Protein Has Distinct m7G Cap- and RNA-binding Sites

Hantaviruses, members of the Bunyaviridae family, are emerging category A pathogens that carry three negative stranded RNA molecules as their genome. Hantavirus nucleocapsid protein (N) is encoded by the smallest S segment genomic RNA (viral RNA). N specifically binds mRNA caps and requires four nucleotides adjacent to the cap for high affinity binding. We show that the N peptide has distinct cap- and RNA-binding sites that independently interact with mRNA cap and viral genomic RNA, respectively. In addition, N can simultaneously bind with both mRNA cap and vRNA. N undergoes distinct conformational changes after binding with either mRNA cap or vRNA or both mRNA cap and vRNA simultaneously. Hantavirus RNA-dependent RNA polymerase (RdRp) uses a capped RNA primer for transcription initiation. The capped RNA primer is generated from host cell mRNA by the cap-snatching mechanism and is supposed to anneal with the 3' terminus of vRNA template during transcription initiation by single G-C base pairing. We show that the capped RNA primer binds at the cap-binding site and induces a conformational change in N. The conformationally altered N with a capped primer loaded at the cap-binding site specifically binds the conserved 3' nine nucleotides of vRNA and assists the bound primer to anneal at the 3' terminus. We suggest that the cap-binding site of N, in conjunction with RdRp, plays a key role during the transcription and replication initiation of vRNA genome.

Off-Target Analysis of Control siRNA Molecules Reveals Important Differences in the Cytokine Profile and Inflammation Response of Human Fibroblasts

The use of RNA interference for the manipulation of gene expression has seen great applications from basic science to clinical investigations. However, limited selectivity and the induction of off-target effects by double stranded RNA molecules have been analyzed and discussed since the discovery of this gene expression regulation mechanism. In this study, the specificity of 13 commercially available control siRNA molecules is addressed by the analysis of gene expression profiles in 2 human cell lines HT1080 and HaCaT and in the mouse cell line 3T3-L1. The off-target signatures of the transfected siRNA molecules differ greatly between the cell lines and only a small overlap was seen for the 2 human cell lines. In particular, the HT1080 cell line showed the highest number of detected gene expression differences. In these cells, several different control siRNA molecules activated a common profile of 79 deregulated genes including a reduced interleukin-1beta (IL-1beta) and IL-24 expression. Functional analysis of MMP1 secretion and tumor necrosis factor-alpha (TNF-alpha) induced IL-8 release revealed a reduction of NFkappaB signaling caused by at least 2 out of the 13 tested control siRNA molecules. Our findings strongly argue for a careful analysis of the control siRNA molecules for any given RNAi experiment.

Measuring Direct Forces on dsRNA in Solid State Nanopores

In recent years, far-reaching discoveries about the functionality of RNA in biology have been made. Especially double stranded RNA (dsRNA) is found to play a key role in the process of RNA interference. We employ solid state nanopores (nanometer sized holes in a thin SiN membrane) to study single RNA molecules. By applying an electrical field over the nanopore, RNA molecules can be threaded into the nanopore, causing a change in the ionic current. This change can provide insight into some of their structural properties, such as charge density, diameter, and possibly also their local structure. We have integrated our nanopore setup with optical tweezers, which allows us to also measure and apply forces to the molecule inside the nanopore.Here, we present the first application of this new technique to the study of RNA molecules, in this case long dsRNA. We show that the force experienced on these molecules is very similar to that on DNA molecules, as one would expect from the very similar structure of these molecules. In addition, we show that the measured force is independent on the distance of the optical trap to the nanopore, even at very close range (< 500 nm). Measuring forces at such close distances may be required for the application of this technique to more complicated molecules, such as single stranded RNA molecules or RNA-protein complexes. Finally, we have further extended the use of this technique to very small nanopores (down to ∼3 nm in diameter), also an important future requirement to study more complex molecules. Combined, these measurements represent important steps towards the detection of local structure along RNA molecules.

Performance and Evaluation of MicroRNA Gene Identification Tools

MicroRNAs are small single stranded RNA molecules of ~ 22 nt in length which play important role in post transcriptional gene regulation either by translational repression of mRNA or by their cleavage. Since their discovery, continuous efforts to identify the miRNA genes led to the discovery of several miRNAs in plants as well as animals. Owing to the limitations of the molecular genetic techniques of miRNA identification, computational approaches were introduced for better and affordable in silico-miRNA predictions. Here, we compared a few miRNA gene identification tools, such as ‘MiPred’,‘Triplet-SVM’,‘BayesMiRNAfind’,‘OneClassmiRNAfind’and ‘BayesSVMmiRNAfind’ to evaluate the performance of its predictability based on the real and pseudo precursor miRNA datasets. Of all the tools examined MiPred is more sensitive (96%) in identifying pseudo miRNAs than Triplet-SVM for real/pseudo miRNA classification, whereas for mature miRNA prediction ‘one-class’ SVM classifier shows best specificity (96%), while BayesSVMmiRNAfind shows least specificity (8%).

English

RNA interference (RNAi) is a naturally occurring phenomenon of RNA-mediated gene silencing that is highly conserved among multicellular organisms. In the first step of the pathway, long double- stranded RNA molecules are chopped into shorter duplexes with 2 nucleotide overhangs at both 3 ' ends by an endonuclease dubbed Dicer, the structure of which has been solved only recently. This results in the formation of small 21 nucleotide long RNAs, aptly named small or short interfering RNAs (siRNAs), which are incorporated into a multimeric protein complex, the RNA-induced silencing complex (RISC). One of the two-siRNA strands guides RISC to a complementary RNA. After hybridization the endonucleolytic slicer activity of RISC cleaves the target RNA, thus preventing its translation. While long double-stranded RNA molecules can be employed to induce RNAi in lower eukaryotes, siRNAs being 21 nucleotides in length have to be used for gene silencing in mammalian cells in order to prevent the activation of an unspecific interferon response (1). In contrast to siRNAs, however, miRNAs are capable of inhibiting translation of the targeted mRNA without degrading it (at least in mammalian cells)(2-4). The need for in silico analysis of the components of the RNA interference pathway arises from the fact that very little is known about the structural and interacting properties of the components. With the above background the analysis was performed to identify putative catalytic motifs in the mRNA of the DICER enzyme.