UTR Assay Research Articles

MiR-1246 in tumor extracellular vesicles promotes metastasis via increased tumor cell adhesion and endothelial cell barrier destruction.

Tumor blood vessels play a key role in tumor metastasis. We have previously reported that tumor endothelial cells (TECs) exhibit abnormalities compared to normal endothelial cells. However, it is unclear how TECs acquire these abnormalities. Tumor cells secrete extracellular vesicles (EVs) to create a suitable environment for themselves. We have previously identified miR-1246 to be more abundant in high metastatic melanoma EVs than in low metastatic melanoma EVs. In the current study, we focused on miR-1246 as primarily responsible for acquiring abnormalities in TECs and examined whether the alteration of endothelial cell (EC) character by miR-1246 promotes cancer metastasis. We analyzed the effect of miR-1246 in metastatic melanoma, A375SM-EVs, in vivo metastasis. The role of tumor EV-miR-1246 in the adhesion between ECs and tumor cells and the EC barrier was addressed. Changes in the expression of adhesion molecule and endothelial permeability were examined. Intravenous administration of A375SM-EVs induced tumor cell colonization in the lung resulting in lung metastasis. In contrast, miR-1246 knockdown in A375SM decreased lung metastasis in vivo. miR-1246 transfection in ECs increased the expression of adhesion molecule ICAM-1 via activation of STAT3, followed by increased tumor cell adhesion to ECs. Furthermore, the expression of VE-Cadherin was downregulated in miR-1246 overexpressed EC. A375SM-EV treatment enhanced endothelial permeability. VE-Cadherin was validated as the potential target gene of miR-1246 via the target gene prediction database and 3' UTR assay. miR-1246 in high metastatic tumor EVs promotes lung metastasis by inducing the adhesion of tumor cells to ECs and destroying the EC barrier.

Abstract 3757: miR-200 inhibits the activation of cancer-associated fibroblasts in lung tumor microenvironment

Abstract Cancer-associated fibroblasts (CAFs) are key components of the tumor microenvironment and promote cancer progression and metastasis. CAFs are regulated by diverse processes including regulation by miRNAs. To identify miRNAs involved in the regulation of CAF activation, we profiled miRNA expression within mouse lung normal fibroblasts (LFs) and CAFs using the NanoString nCounter method. Among numerous miRNAs differentially expressed between CAFs and LFs, miR-200 was selected for further investigation, which is more highly downregulated in CAFs. Overexpression of miR-200 in CAFs suppressed the migration and invasion of lung cancer cells in co-culture systems and inhibited metastasis in a mouse model. In addition, we verified that lung cancer patients with high miR-200 levels showed a good prognosis than those with low miR-200 levels. Next, we tried to identify novel miR-200 targets based on RNA sequencing data, lung adenocarcinoma TCGA, and TargetScan. NRP2 was confirmed to be a target of miR-200 by 3΄-UTR assay. NRP2 knockdown by siRNAs in CAFs suppressed cancer cell invasion, and NRP2 addback in CAF-200 restored cancer cell invasion inhibited by miR-200. NRP2 functions as a co-receptor with VEGFR, thus, CAF-200 was less responsive to VEGF than control CAFs. Collectively, this study suggests that downregulation of miR-200 in CAFs promotes lung cancer progression in tumor microenvironment; therefore, miR-200 will be a good therapeutic target or a biomarker in lung adenocarcinoma. Citation Format: Inyoung Cheon, Sieun Lee. miR-200 inhibits the activation of cancer-associated fibroblasts in lung tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3757.

MiR-130b modulates the invasive, migratory, and metastatic behavior of leiomyosarcoma.

Leiomyosarcoma (LMS) is an aggressive, often poorly differentiated cancer of the smooth muscle (SM) lineage for which the molecular drivers of transformation and progression are poorly understood. In microRNA (miRNA) profiling studies, miR-130b was previously found to be upregulated in LMS vs. normal SM, and down-regulated during the differentiation of mesenchymal stem cells (MSCs) into SM, suggesting a role in LMS tumor progression. In the present study, the effects of miR-130b on human LMS tumorigenesis were investigated. Stable miR-130b overexpression enhanced invasion of LMS cells in vitro, and led to the formation of undifferentiated, pleomorphic tumors in vivo, with increased growth and metastatic potential compared to control LMS cells. TSC1 was identified as a direct miR-130b target in luciferase-3'UTR assays, and shRNA-mediated knockdown of TSC1 replicated miR-130b effects. Loss-of-function and gain-of-function studies showed that miR-130b levels regulate cell morphology and motility. Following miR-130b suppression, LMS cells adopted a rounded morphology, amoeboid mode of cell movement and enhanced invasive capacity that was Rho/ROCK dependent. Conversely, miR-130b-overexpressing LMS cells exhibited Rho-independent invasion, accompanied by down-regulation of Rho-pathway effectors. In mesenchymal stem cells, both miR-130b overexpression and TSC1 silencing independently impaired SM differentiation in vitro. Together, the data reveal miR-130b as a pro-oncogenic miRNA in LMS and support a miR-130b-TSC1 regulatory network that enhances tumor progression via inhibition of SM differentiation.

Effect of MiR-100-5p on proliferation and apoptosis of goat endometrial stromal cell in vitro and embryo implantation in vivo.

The growth of endometrial stromal cells (ESCs) at implantation sites may be a potential factor affecting the success rate of embryo implantation. Incremental proofs demonstrated that ncRNAs (e.g. miRNAs, lncRNAs and circRNAs) were involved in various biological procedures, including proliferation and apoptosis. In this study, the role of miR‐100‐5p on proliferation and apoptosis of goat ESCs in vitro and embryo implantation in vivo was determined. The mRNA expression of miR‐100‐5p was significantly inhibited in the receptive phase (RE) rather than in the pre‐receptive phase (PE). Overexpression of miR‐100‐5p suppressed ESCs proliferation and induced apoptosis. The molecular target of MiR‐100‐5p, HOXA1, was confirmed by 3′‐UTR assays. Meanwhile, the product of HOXA1 mRNA RT‐PCR increased in the RE more than that in the PE. The HOXA1‐siRNA exerted significant negative effects on growth arrest. Instead, incubation of ESCs with miR‐100‐5p inhibitor or overexpressed HOXA1 promoted the cell proliferation. In addition, Circ‐9110 which acted as a sponge for miR‐100‐5p reversed the relevant biological effects of miR‐100‐5p. The intrinsic apoptosis pathway was suppressed in ESCs, revealing a crosstalk between Circ‐9110/miR‐100‐5p/HOXA1 axis, PI3K/AKT/mTOR, and ERK1/2 pathways. To further evaluate the progress in study on embryo implantation regulating mechanism of miR‐100‐5p in vivo, the pinopodes of two phases were observed and analysed, suggesting that, as similar as in situ, miR‐100‐5p was involved in significantly regulating embryo implantation in vivo. Mechanistically, miR‐100‐5p performed its embryo implantation function through regulation of PI3K/AKT/mTOR and ERK1/2 pathways by targeting Circ‐9110/miR‐100‐5p/HOXA1 axis in vivo.

Isolation and Genomic Characterization of Echovirus 11 from faeces of a Non-Human Primate in Nigeria.

We recently investigated the presence of enteroviruses (EVs) in non-human primates (NHPs) in Northern Nigeria and documented the presence of EV-A76 of South-East Asian ancestry in an NHP. In this study, we go further to ask if we could also find EVs in NHPs indigenous to the forested South-south Nigeria. Fresh faecal samples were collected from the floor of 10 cages housing NHPs in Cross River Nigeria, re-suspended in PBS and subjected to RNA extraction, cDNA synthesis, PanEnt 5'-UTR and PanEnt VP1 PCR assays. None of the samples was positive for the PanEnt VP1 assay, but one sample was positive for PanEnt 5'-UTR PCR. This sample was subsequently inoculated into RD cell line, produced CPE and the isolate analysed by PCR assays, next-generation whole genome sequencing and passage in four different cell lines showing replication in two of them. Analysis of the complete genome of the isolate identified it as an Echovirus 11 (E11) and revealed a recombinant genomic structure. Phylogenetic analysis showed that the E11 NHP strain was related to human clinical isolates suggesting a zoonotic behaviour. We describe the first isolation and complete genome characterization of an E11 obtained from an NHP in Nigeria having zoonotic potential.

Full sequence of mutant huntingtin 3'-untranslated region and modulation of its gene regulatory activity by endogenous microRNA.

Huntington's disease (HD) is caused by an expanded CAG trinucleotide repeat in the first exon of the huntingtin gene (HTT). Since the entire course of the disease starts from this dominant gain-of-function mutation, lowering total or mutant huntingtin mRNA/protein has emerged as an appealing therapeutic strategy. We reasoned that endogenous mechanisms underlying HTT gene regulation may inform strategies to target the source of the disease. As part of our investigation to understand how the expression of HTT is controlled, we performed (1) complete sequencing analysis for mutant HTT 3'-UTR and (2) unbiased screening assays to identify naturally-occurring miRNAs that could lower the HTT mRNA levels. By sequencing HD families inheriting the major European mutant haplotype, we determined the full sequence of HTT 3'-UTRs of the most frequent mutant (i.e., hap.01) and normal (i.e., hap.08) haplotypes, revealing 5 sites with alternative alleles. In subsequent miRNA activity assays using the full-length hap.01 and hap.08 3'-UTR reporter vectors and follow-up validation experiments, hsa-miR-4324 and hsa-miR-4756-5p significantly reduced HTT 3'-UTR reporter activity and endogenous HTT protein levels. However, those miRNAs did not show strong haplotype-specific effects. Nevertheless, our data highlighting full sequences of HTT 3'-UTR haplotypes, effects of miRNAs on HTT levels, and potential interaction sites provide rationale and promising targets for total and mutant-specific HTT lowering intervention strategies using endogenous and artificial miRNAs, respectively.

MicroRNAs are Critical Regulators of Osteoclast Differentiation.

Our goal is to comprehensively review the most recent reports of microRNA (miRNA) regulation of osteoclastogenesis. We highlight validated miRNA-target interactions and their place in the signaling networks controlling osteoclast differentiation and function. Using unbiased approaches to identify miRNAs of interest and reporter-3'UTR assays to validate interactions, recent studies have elucidated the impact of specific miRNA-mRNA interactions during in vitro osteoclastogenesis. There has been a focus on signaling mediators downstream of the RANK and CSF1R signaling, and genes essential for differentiation and function. For example, several miRNAs directly and indirectly target the master osteoclast transcription factor, Nfatc1 (e.g. miR-124 and miR-214) and Rho-GTPases, Cdc42 and Rac1 (e.g. miR-29 family). Validating miRNA expression patterns, targets, and impact in osteoclasts and other skeletal cells is critical for understanding basic bone biology and for fulfilling the therapeutic potential of miRNA-based strategies in the treatment bone diseases.

MicroRNAs-361-5p and miR-574-5p associate with human adipose morphology and regulate EBF1 expression in white adipose tissue.

Reduced adipose expression of the transcription factor Early B cell factor 1 (EBF1) is linked to white adipose tissue (WAT) hypertrophy. We aimed to identify microRNAs (miRNAs) associated with WAT hypertrophy and EBF1 regulation. We mapped WAT miRNA expression from 26 non-obese women discordant in WAT morphology and determined EBF1 activity in the non-obese and 30 obese women. Expression of 15 miRNAs was higher in hypertrophy and 10 were predicted to target EBF1. Binding of miR-365-5p/miR-574-5p were validated with 3'-UTR assay. Overexpression of miR-365-5p or miR-574-5p reduced EBF1 while inhibition of miR-574 increased EBF1 expression in human adipocytes invitro. Additive effects on EBF1 were observed when concomitantly overexpressing both miRNAs. EBF1 targets were affected by over expression/inhibition of either miRNAs. Finally, miR-365-5p/miR-574-5p expression in 56 individuals correlated significantly with EBF1 activity. Our results suggest that miR-365-5p and miR-574-5p may be linked to WAT hypertrophy via effects on EBF1 expression.

Abstract B23: Role of noncoding RNAs in regulating human papilloma virus-16 (HPV-16) induced oncogenesis in head and neck squamous cell carcinoma (HNSCC)

Abstract Background: HPV is an emerging risk factor for HNSCC, however, molecular mechanisms in HPV mediated HNSCC remains poorly delineated. Recent studies suggest that non-coding RNAs may play an important role in the aetiology of HNSCC. In the present study, we examined microRNAs involved in HPV-16 induced HNSCC. Material and Method: Tissue samples from HNSCC patients (n=300) were screened for HPV-16 using PCR based approach with consensus PCR primers (MY09/MY11, Gp5+/Gp6+), HPV16-specific PCR, HPV-16 E7 mRNA, immunohistochemistry (p16, p53 and pRB) and viral-load determination. In-silico approach was used to identify host miRNAs with HPV-16 mRNAs as putative target. Differential expression of identified miRNAs was determined by qRT-PCR in HPV-16 positive (UPCI:SCC090, CaSki, SiHa), HPV negative (UPCI:SCC116, HaCat) and transfected with HPV 16 full genome plasmid cell lines, and, HPV 16-positive/HPV-negative tissue samples. Putative targets of identified miRNAs were identified by bioinformatic approach and their mRNA levels were determined by qRT-PCR in HPV positive and negative cell lines, and tissue samples. Over-expression and inhibition studies with miRNA mimic and inhibitor were done to identify affected viral and host oncogenic pathways. Results: Total 32 HNSCC patients were found to be HPV-16 positive. We identified 10 miRNAs with HPV-16 as putative target. Among them hsa-miR-139-3p showed significant down-regulation in HPV-16 positive samples and cell lines compared to HPV negative controls. Using bioinformatics tools, hsa-miR-139-3p was found to target viral HPV-16 E1 and host HOXB6 gene. Transfection with hsa-miR-139-3p mimic resulted in significant down-regulation of HPV 16 early mRNA as well as its targeted genes in luciferase-3'UTR assay. Moreover, mimic transfected HPV-positive cell lines showed dysregulated MEK-ERK signaling pathway. Conclusion: Our results suggest hsa-miR-139-3p may be involved in patho-physiology of HPV-16 positive HNSCC as it deregulates expression of HPV oncogenic proteins and cell proliferation by targeting MEK-ERK signaling pathway. Citation Format: Malay Kumar Sannigrahi, Varinder Singh, Rajni Sharma, Naresh K. Panda, Madhu Khullar. Role of noncoding RNAs in regulating human papilloma virus-16 (HPV-16) induced oncogenesis in head and neck squamous cell carcinoma (HNSCC). [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B23.

MiR130b-Regulation of PPARγ Coactivator- 1α Suppresses Fat Metabolism in Goat Mammary Epithelial Cells.

Fat metabolism is a complicated process regulated by a series of factors. microRNAs (miRNAs) are a class of negative regulator of proteins and play crucial roles in many biological processes; including fat metabolism. Although there have been some researches indicating that miRNAs could influence the milk fat metabolism through targeting some factors, little is known about the effect of miRNAs on goat milk fat metabolism. Here we utilized an improved miRNA detection assay, S-Poly-(T), to profile the expression of miRNAs in the goat mammary gland in different periods, and found that miR-130b was abundantly and differentially expressed in goat mammary gland. Additionally, overexpressing miR-130b impaired adipogenesis while inhibiting miR-130b enhanced adipogenesis in goat mammary epithelial cells. Utilizing 3’-UTR assay and Western Blot analusis, the protein peroxisome proliferator-activated receptor coactivator-1α (PGC1α), a major regulator of fat metabolism, was demonstrated to be a potential target of miR-130b. Interestingly, miR-130b potently repressed PGC1α expression by targeting both the PGC1α mRNA coding and 3’ untranslated regions. These findings have some insight of miR-130b in mediating adipocyte differentiation by repressing PGC1α expression and this contributes to further understanding about the functional significance of miRNAs in milk fat synthesis.

Abstract 2306: MicroRNA 199a*: a potent suppressor of tumor metastasis and angiogenesis.

Abstract The growth of a solid tumor depends on diffusion of nutrients from the tumor microenvironment through vascular system. Angiogenesis, the new blood vessels formation is the primary route by which tumor cells get nutrients as well as exit from primary site and enter into the blood circulation. The proliferation and migration of vascular endothelial cells are triggered by angiogenic growth factors secreted by tumor cells, such as VEGF, TGF-β etc. Recent evidences showed HGF also plays an important role in angiogenesis by enhancing the proliferative activity and intracellular signaling. HGF/its receptor, c-MET and VEGF/VEGFR1 are two independent pathways for angiogenesis and metastasis. Thus detection of a molecule that hits both pathways could be a potent therapeutic agent that could facilitate the inhibition of improved barrier function. Recently mir199a* has been shown to target both CD44 and cMET molecule to block the metastasis in cancer. The present study aim to show that mir199a* also target HGF, the key modulator of the HGF/c-Met signaling cascade in epithelial cells to shut off the mesenchymal transition and VEGFR1 to prevent angiogenesis more effectively. Luciferase 3’ UTR assay was performed to investigate HGF as a target of mir-199a*. HGF mRNA and protein expression was determined by real time RT-PCR, Western blot analysis in liver stellate cell line LX2 transfected with mir-199a* and HGF shRNA. The cultured media of mir-199a* transfected LX2 cells was used to verify the effect of mir-199a* on HGF induced migration and invasion ability on metastatic cell lines using Boyden chamber assay with or without matrigel coated membrane and angiogenesis by tube formation assay. The mir-199a* binding site was detected by pictar, target scan analysis of the 3’UTR of HGF and VEGFRs. The binding of mir-199a* to the 3′UTR of HGF and VEGFR1 were confirmed by luciferase assay, mutagenesis and western blotting. Down regulation of HGF protein expression in HGF producing hepatic stellate cell, LX2 was observed by mir-199a*. Addition of LX2 conditioned media transfected with mir199a* to metastatic cell reduces the migration and invasion and also block angiogenesis in endothelial cells. As Mir-199a* could target HGF/cMET, CD44-cMET and VEGF/VEGFR1 signaling cascade, it could be used as a potential therapeutic target of HCC. Citation Format: Alip Ghosh, Amit Ghosh, Debanjali Dasgupta, Shrabasti Roychowdhury, Shrabasti Roychowdhury, Simanti Datta, Abhijit Chowdhury, Soma Banerjee. MicroRNA 199a*: a potent suppressor of tumor metastasis and angiogenesis. [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 2306. doi:10.1158/1538-7445.AM2013-2306

MiR‐197 induces epithelial–mesenchymal transition in pancreatic cancer cells by targeting p120 catenin

Invasive ductal adenocarcinoma (IDA) of the pancreas manifests poor prognosis due to the early invasion and distant metastasis. In contrast, intraductal papillary mucinous adenoma or carcinoma (IPMA or IPMC) reveals better clinical outcomes. Various molecular mechanisms contribute to these differences but entire picture is still unclear. Recent researches emphasized the important role of miRNA in biological processes including cancer invasion and metastasis. We previously described that miR-126 is down-regulated in IDA compared with IPMA or IPMC, and miR-126 regulates the expression of invasion related molecule disintegrin and metalloproteinase domain-containing protein 9 (ADAM9). Assessing the difference of miRNA expression profiles of IDA, IPMA, and IPMC, we newly identified miR-197 as an up-regulated miRNA specifically in IDA. Expression of miR-197 in pancreatic cancer cells resulted in the induction of epithelial-mesenchymal transition (EMT) along with the down-regulation of p120 catenin which is a putative target of miR-197. Direct interaction between miR-197 and p120 catenin mRNA sequence was confirmed by 3'UTR assay, and knockdown of p120 catenin recapitulated EMT induction in pancreatic cancer cells. In situ hybridization of miR-197 and immunohistochemistry of p120 catenin showed mutually exclusive patterns suggesting pivotal role of miR-197 in the regulation of p120 catenin. This miR-197/p120 catenin axis could be a novel therapeutic target.

MiR-125b, a Target of CDX2, Regulates Cell Differentiation through Repression of the Core Binding Factor in Hematopoietic Malignancies

MicroRNA-125b (miR-125b), a small noncoding RNA molecule, has been found to be deregulated and functions as an oncogene in many cancers including hematopoietic malignancies. However, the mechanisms accounting for miR-125b dysregulation remain to be elucidated. The present study aims to identify the factors that might contribute to up-regulation of miR-125b in human hematopoietic malignancies and its downstream targets for lineage-specific differentiation. We at first reported that CDX2, a homeobox transcription factor, binds to promoter regions of the miR-125b gene and activates transcriptional regulation of miR-125b in malignant myeloid cells. We further revealed that increasing levels of CDX2 in malignant myeloid cells activate miR-125b expression, which in turn inhibits core binding factor β (CBFβ) translation, thereby counteracting myeloid cell differentiation, at least for granulocytic lineage, and promoting leukemogenesis. Interestingly, we found that this novel pathway including CDX2, miR-125b, and CBFβ was mediated by undergoing all-trans-retinoic acid induction. Once differentiation ensues with all-trans-retinoic acid treatment, CDX2 activity decreases, leading to a reduction in miR-125b transcription and up-regulation of CBFβ in myeloid cells and in patients. The study provides a new mechanism that contributes to hematopoietic malignancies, which could involve deregulation of miR-125b and its up- and downstream factors. As altered expression of miRNAs has been reported in a wide range of malignancies, delineating the underlying molecular mechanisms of aberrant miRNA expression and characterizing the upstream and downstream factors will help to understand important steps in the pathogenesis of these afflictions.

Isoform-level microRNA-155 target prediction using RNA-seq

Computational prediction of microRNA targets remains a challenging problem. The existing rule-based, data-driven and expression profiling approaches to target prediction are mostly approached from the gene-level. The increasing availability of RNA-seq data provides a new perspective for microRNA target prediction on the isoform-level. We hypothesize that the splicing isoform is the ultimate effector in microRNA targeting and that the proposed isoform-level approach is capable of predicting non-dominant isoform targets as well as their targeting regions that are otherwise invisible to many existing approaches. To test the hypothesis, we used an iterative expectation maximization (EM) algorithm to quantify transcriptomes at the isoform-level. The performance of the EM algorithm in transcriptome quantification was examined in simulation studies using FluxSimulator. We used joint evidence from isoform-level down-regulation and seed enrichment to predict microRNA-155 targets. We validated our computational approach using results from 149 in-house performed in vitro 3′-UTR assays. We also augmented the splicing database using exon–exon junction evidence, and applied the EM algorithm to predict and quantify 1572 cell line specific novel isoforms. Combined with seed enrichment analysis, we predicted 51 novel microRNA-155 isoform targets. Our work is among the first computational studies advocating the isoform-level microRNA target prediction.

Abstract 2072: Systematic functional analysis of microRNAs by transfection of 1129 miRNAs into prostate cancer cells

Abstract The molecular mechanisms behind prostate cancer development, progression and therapy failure are incompletely understood, but the androgen receptor (AR) is almost invariably involved. Altered microRNA (miRNA) expression profiles have recently been demonstrated in a large number of cancer types, including prostate cancer. These short RNA molecules of ∼21 bases regulate mRNA stability and translation by base-pairing mainly to the 3’-untranslated region (3′UTR) of their targets. In this study, we analyzed the functional impact of miRNA transfections on prostate cancer cell growth, survival, and expression of the AR protein. Seven prostate cell lines (LNCaP, LAPC-4, MDA-PCa-2b, 22Rv1, CWR-1R, RWPE-1, and EP156T) were screened with 1129 miRNA mimics (Ambion, Dharmacon). Cell viability was measured with CellTiter-Glo® (CTG, Promega) whereas AR, cell proliferation (Ki67) and apoptosis (cPARP) were detected with specific antibodies using protein lysate microarrays as described in our recent publication on estrogen receptor targeting miRNAs (Leivonen et al., Oncogene 2009). Seventy-one of the miRNAs had anti-proliferative or pro-apoptotic functions when transfected to the prostate cancer cell lines. In addition, 17 of these miRNAs down-regulated the AR protein. Western blots were used to verify knockdown, and the direct functional role of the miRNAs was clarified by 3′UTR assays and by analyses of down-stream consequences of the knockdowns. In summary, we have systematically defined those miRNAs that are critical for prostate cancer growth, survival and the expression of the androgen receptor. This integrated, multi-parametric profiling provides new evidence of the role of miRNAs in prostate cancer as well as novel clues for therapeutic targeting by manipulating key pathways and cell survival mechanisms by miRNA transfections. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 2072.

Fluorescence-based multiplex real-time RT-PCR arrays for the detection and serotype determination of foot-and-mouth disease virus

This study reports the development of distinct fluorescence-based multiplex rRT-PCR arrays, which are comprised of unique primer pairs that are statistically modeled from publicly available sequences, for overall foot-and-mouth disease virus and serotype-specific detection. Performance of the multiplex pan FMDV (MpFMDV) array was compared to the 5′ UTR and two 3D pol assays, following tests on the Miniopticon (BioRad) and 7900HT SDS (ABI) platforms. The MpFMDV array and a recently developed alternative 3D pol assay correctly identified all 31 isolates that represented all seven serotypes, including 10 isolates reported previously to be undetectable by both OIE recommended assays (5′ UTR and 3D pol assay). Statistically significant differences in mean C T values were observed with both platforms. With the Miniopticon, the MpFMDV assay detected FMDV at 9.86 and 2.67 cycles less than the 5′ UTR and one of the 3D pol assay, respectively; whereas with the 7900HT SDS, the MpFMDV assay detected FMDV at 7.11, 4.71, and 2.33 cycles less than the 5′ UTR and both 3D pol assays, respectively. The MpFMDV assay was more sensitive than the 5′ UTR assay, which had a higher mean endpoint dilution of 1.3 log 10. With the exception of the serotype A and C multiplex arrays, the multiplex serotype-specific arrays correctly identified all isolates from five of seven serotypes (O, Asia 1, SAT 1, 2, and 3). Results presented in this study provide proof-of-principle and bench validation for the primer design model, and for the ability of multiplex arrays to accurately detect FMDV and to provide some degree of serotype discrimination of FMDV.

Discordant results from hepatitis C virus genotyping by procedures based on amplification of different genomic regions

We compared the results of genotyping hepatitis C virus (HCV) either by PCR amplification of the core region or by hybridization of PCR-amplified products of the 5' untranslated region (5'UTR assay). Serum samples from 144 Italian anti-HCV-positive patients (106 drug abusers and 38 patients with chronic viral liver disease but no history of drug abuse) were studied. The original core region assay described by Okamoto et al. (H. Y. Okamoto, Y. Sugiyama, S. Okada, K. Kurai, Y. Akahane, Y. Sugai, T. Tanaka, K. Sato, F. Tsuda, Y. Miyakawa, and M. Mayumi, J. Gen. Virol. 73:673-679, 1992) allowed genotyping of 75 of 144 samples. A modified version of Widell et al. (A. Widell, S. Shev, S. Månsson, Y.-Y. Zhang, U. Foberg, G. Norkrans, A. Frydén, O. Weiland, J. Kurkus, and E. Nordenfelt, J. Med. Virol. 44:272-279, 1994) allowed genotyping of 11 of 79 samples (50 of 79 samples remained unclassified by the method of Okamoto et al. In contrast, all 144 samples were genotyped by the 5'UTR assay. Forty-six of 75 (61 percent) of the samples genotyped by the method of Okamoto et al. and 10 of 11 (91 percent) of the samples genotyped by the method of Widell et al. had results consistent with those obtained by the 5'UTR assay. According to the results of direct sequencing, the method of Okamoto et al. erroneously classified seven samples as having mixed infections. In conclusion, HCV genotyping seems more reliable when it is performed by the 5'UTR assay than by either of two core region assays. The major advantage provided by the 5'UTR assay is a much lower proportion of negative or indeterminate results in younger patients with histories of drug abuse or infection by genotypes other than HCV type 1.