Human microRNA Expression Research Articles

Regulation of microRNA with food. Part 1. Food of plant origin

Тhe scientific review presents the mechanisms of microRNA regulation of biological processes in the human body with the help of food products of plant origin. To write the article, information was searched using Scopus, Web of Science, MedLine, PubMed, Google Scholar, EMBASE, Global Health, The Cochrane Library databases. The obtained information on dietary microRNAs is organized into the Dietary MicroRNA Databases (http://sbbi.unl.edu/dmd/), which is a repository for archiving and analyzing structural and functional microRNAs that enter the human body with food. It is stated that exogenous plant miRNAs entering the body with food are present in blood serum, tissues of humans and various animals, and regulate the expression of target genes. More than 50 miRNAs of plant origin have been identified in human blood serum using next-generation sequencing. The authors list some representatives from the multitude of dietary plant miRNAs. It is emphasized that several miRNAs show their functional activity both in plants and in mammals and easily overcome physiological barriers (miR-155, miR-168 and miR-854). Scientists believe that the ambiguous effect of plant products on the expression of human microRNA was shown by the results of a study conducted in vegetarians. Thus, with a comprehensive review using the latest information search databases, it was found that in modern scientific literature, the authors determine the horizontal transfer of numerous microRNA molecules from plants to the human body. Exogenous xenogenic xenomiRs are identified in most samples of human tissues and biological fluids. Entering the body together with food, they can significantly change the structure of the human transcriptome. Food products not only bring xenogeneic miRNAs, but also act as modulators of the endogenous miRNA generation by cells of the human body.

Effect of Chronic Radiation Exposure on Human MicroRNA Expression

Effect of Chronic Radiation Exposure on Human MicroRNA Expression

Effect of Chronic Radiation Exposure on Human microRNA Expression

Relative expression of mature microRNA assessed in peripheral blood cells in persons chronically exposed to low-dose radiation in the long-term period. The study included people exposed in the 1950s on the Techa River (Southern Urals, Russia). The cumulative red bone marrow doses (RBM) of the persons in the main study group (33 persons) ranged from 77.7 to 2869.8 mGy (mean value – 698.5 mGy). The comparison group consisted of 30 people with RBM dose not exceeding 70 mGy over their lifetime. Reverse transcription of RNA samples was performed using specific stem-loop primers (“stem-loop”). Changes in the relative content of microRNA were assessed by real-time PCR on the CFX96 detection system (BioRad, USA). Statistical analysis of the results was performed using Mann–Whitney U-criterion. A significant increase in hsa-miR-125b, hsa-miR-181a, and hsa-miR-16-5p content was detected in exposed people after 60 years, and their expression was found to depend on the RBM dose.

Analysis of Human microRNA Expression Profiling During Diquat-Induced Renal Proximal Tubular Epithelial Cell Injury

Analysis of Human microRNA Expression Profiling During Diquat-Induced Renal Proximal Tubular Epithelial Cell Injury

JC Polyomavirus Modifies the Expression of Human microRNAs in Progressive Multifocal Leukoencephalopathy Brain.

Progressive multifocal leukoencephalopathy (PML) is a severe neurological condition caused by reactivation of JC polyomavirus (JCPyV) in immunosuppression. Asymptomatic JCPyV persists in peripheral tissues. Upon reactivation, neurotropic rearrangements may emerge, and the virus gains access to the brain. To assess the mechanisms of PML pathogenesis, brain tissue material from PML patients was collected for small RNA sequencing. Upregulation of 8 microRNAs (miRNAs) in PML brain was validated using quantitative microRNA polymerase chain reaction (PCR). Bioinformatics tools were utilized to identify major associations of the upregulated miRNAs: neuroinflammation and blood-brain barrier disruption. The results indicate involvement of human miRNA regulation in PML pathogenesis.

Differential Expression of Human MicroRNAs During Dengue Virus Infection in THP-1 Monocytes.

Dengue virus (DENV) is the most widespread arbovirus, responsible for a wide range of clinical manifestations, varying from self-limited illness to severe hemorrhagic fever. Dengue severity is associated with host intense proinflammatory response and monocytes have been considered one of the key cell types involved in the early steps of DENV infection and immunopathogenesis. To better understand cellular mechanisms involved in monocyte infection by DENV, we analyzed the expression levels of 754 human microRNAs in DENV-infected THP-1 cells, a human monocytic cell line. Eleven human microRNAs showed differential expression after DENV infection and gene ontology and enrichment analysis revealed biological processes potentially affected by these molecules. Five downregulated microRNAs were significantly linked to cellular response to stress, four to cell death/apoptosis, two to innate immune responses and one upregulated to vesicle mediated, TGF-β signaling, phosphatidylinositol mediated signaling, lipid metabolism process and blood coagulation.

Cellular and Molecular Response of Macrophages THP-1 during Co-Culture with Inactive Trichophyton rubrum Conidia.

Trichophyton rubrum is causing an increasing number of invasive infections, especially in immunocompromised and diabetic patients. The fungal invasive infectious process is complex and has not yet been fully elucidated. Therefore, this study aimed to understand the cellular and molecular mechanisms during the interaction of macrophages and T. rubrum. For this purpose, we used a co-culture of previously germinated and heat-inactivated T. rubrum conidia placed in contact with human macrophages cell line THP-1 for 24 h. This interaction led to a higher level of release of interleukins IL-6, IL-2, nuclear factor kappa beta (NF-κB) and an increase in reactive oxygen species (ROS) production, demonstrating the cellular defense by macrophages against dead fungal elements. Cell viability assays showed that 70% of macrophages remained viable during co-culture. Human microRNA expression is involved in fungal infection and may modulate the immune response. Thus, the macrophage expression profile of microRNAs during co-culture revealed the modulation of 83 microRNAs, with repression of 33 microRNAs and induction of 50 microRNAs. These data were analyzed using bioinformatics analysis programs and the modulation of the expression of some microRNAs was validated by qRT-PCR. In silico analysis showed that the target genes of these microRNAs are related to the inflammatory response, oxidative stress, apoptosis, drug resistance, and cell proliferation.

Music-listening regulates human microRNA expression

ABSTRACT Music-listening and performance have been shown to affect human gene expression. In order to further elucidate the biological basis of the effects of music on the human body, we studied the effects of music-listening on gene regulation by sequencing microRNAs of the listeners (Music Group) and their controls (Control Group) without music exposure. We identified upregulation of six microRNAs (hsa-miR-132-3p, hsa-miR-361-5p, hsa-miR-421, hsa-miR-23a-3p, hsa-miR-23b-3p, hsa-miR-25-3p) and downregulation of two microRNAs (hsa-miR-378a-3p, hsa-miR-16-2-3p) in Music Group with high musical aptitude. Some upregulated microRNAs were reported to be responsive to neuronal activity (miR-132, miR-23a, miR-23b) and modulators of neuronal plasticity, CNS myelination, and cognitive functions like long-term potentiation and memory. miR-132 plays a critical role in regulating TAU protein levels and is important for preventing tau protein aggregation that causes Alzheimer’s disease. miR-132 and DICER, upregulated after music-listening, protect dopaminergic neurons and are important for retaining striatal dopamine levels. Some of the transcriptional regulators (FOS, CREB1, JUN, EGR1, and BDNF) of the upregulated microRNAs were immediate early genes and top candidates associated with musical traits. BDNF and SNCA, co-expressed and upregulated in music-listening and music-performance, are both are activated by GATA2, which is associated with musical aptitude. Several miRNAs were associated with song-learning, singing, and seasonal plasticity networks in songbirds. We did not detect any significant changes in microRNA expressions associated with music education or low musical aptitude. Our data thereby show the importance of inherent musical aptitude for music appreciation and for eliciting the human microRNA response to music-listening.

MiR‑28‑5p inhibits the migration of breastcancer by regulating WSB2.

MicroRNAs (miRNAs or miRs) play an important role in the tumorigenesis and progression of breast cancer. However, the function of miR-28-5p in breast cancer migration has yet to be determined. In the present study, Human MicroRNA Expression Database (HMED) analysis revealed that the expression level of miR-28-5p was significantly lower in breast cancer tissue than in normal breast tissue. Kaplan -Meier plotter (KMPLOT) analysis revealed that the low expression level of miR-28-5p was associated with a poor survival in breast cancer. In addition, reverse transcription-quantitative PCR (RT-qPCR) revealed that the expression of miR-28-5p was significantly lower in breast cancer cell lines compared with that in human mammary epithelial cells (HMECs). Moreover, transfection with miR-28-5p mimics suppressed the migration of MCF-7 cells, whereas an miR-28-5p inhibitor exerted the opposite effect. Gene chip assay identified 648 differentially expressed genes (DEGs) in cells overexpressing miR-28-5p. The DEGs are enriched in the 'focal adhesion' and 'pathway in cancer' pathways. The expression levels of Ras-related protein Rap-1b (RAP1B), WD repeat and SOCS box containing 2 (WSB2) and vascular endothelial growth factor A (VEGFA) were confirmed by RT-qPCR. Furthermore, transfection with miR-28-5p mimics decreased WSB2 expression, whereas the miR-28-5p inhibitor increased the expression of WSB2, at both the transcriptional and translational levels. miR-28-5p targets the 3′UTR of WSB2, and the binding site is conserved in multiple species, with a consensus motif of 5′-AGCUCCUU-3′. Moreover, WSB2 overexpression promoted the migration of MCF-7 cells which had been inhibited by miR-28-5p. UALCAN analysis revealed that WSB2 was significantly upregulated in primary breast tumor tissue, and a high expression level of WSB2 was associated with a poor survival in breast cancer. Furthermore, immunohistochemistry revealed that the expression of WSB2 was markedly higher in breast cancer tissue compared with that in adjacent normal breast tissue. Taken together, the findings of the present study demonstrate that miR-28-5p inhibits the migration of breast cancer cells by regulating WSB2 expression, and the miR-28-5p/WSB2 axis may be a novel therapeutic target in breast cancer.

Correlation Between the Hepatic Expression of Human MicroRNA hsa-miR-125a-5p and the Progression of Fibrosis in Patients With Overt and Occult HBV Infection.

To evaluate the correlation between the hepatic expression pattern of hsa-miR-125a-5p and HBV-DNA and the progression of fibrosis in patients with overt or occult HBV infection. We enrolled all the HBsAg-positive treatment naive patients (overt HBV group) and all the HBsAg-negative patients with hepatocellular carcinoma and with a positive HBV-DNA in their hepatic tissue (occult HBV group), who underwent a diagnostic liver biopsy between April 2007 and April 2015. Tissue concentrations of HBV-DNA and hsa-miR-125a-5p were then analyzed by real-time quantitative PCR. Necroinflammatory activity and fibrosis were evaluated according to the Ishak score. During the study period, we enrolled 64 patients with overt and 10 patients with occult HBV infection. In the overt HBV group, 35 of 64 (54.7%) showed a mild fibrosis (staging 0-2), 17 (26.6%) a moderate fibrosis (staging 3-4), while the remaining 12 (18.7%) had a cirrhosis. All patients in the occult HBV group were cirrhotic. Patients with more advanced fibrosis stage showed a higher mean age when compared with those with mild (p < 0.00001) or moderate fibrosis (p < 0.00001) and were more frequently male than patients with staging 0-2 (p = 0.04). Similarly, patients with occult B infection were older than HBsAg-positive patients. Liver concentrations of miR-125a-5p were significantly higher in patients with cirrhosis (9.75 ± 4.42 AU) when compared with patients with mild (1.39 ± 0.94, p = 0.0002) or moderate fibrosis (2.43 ± 2.18, p = 0.0006) and were moderately higher in occult than in overt HBV infection (p = 0.09). Moreover, we found an inverse correlation, although not statistically significant, between the tissue HBV-DNA levels and the staging of fibrosis. This study suggests a correlation between the tissue expression of hsa-miR-125a-5p and the progression of liver damage in a group of patients with occult or overt HBV infection. If confirmed, these data suggest the hsa-miR-125a-5p may be a novel biomarker of hepatic damage.

Human microRNA expression in sporadic and FAP-associated desmoid tumors and correlation with beta-catenin mutations.

Desmoid tumors (DT) are rare, benign, fibroblastic neoplasm with challenging histological diagnosis. DTs can occur sporadically or associated with the familial adenomatous polyposis coli (FAP). Most sporadic DTs are associated with β-catenin gene (CTNNB1) mutations, while mutated APC gene causes FAP disease. microRNAs (miRNAs) are involved in many human carcinogenesis.The miRNA profile was analyzed by microarray in formalin-fixed, paraffin-embedded (FFPE) specimens of 12 patients (8 sporadic, 4 FAP-associated) and 4 healthy controls. One hundred and one mRNAs resulted dysregulated, of which 98 in sporadic DTs and 8 in FAP-associated DTs, 5 were shared by both tumors. Twenty-six miRNAs were then validated by RT-qPCR in 23 sporadic and 7 FAP-associated DT samples matched with healthy controls. The qPCR method was also used to evaluate the CTNNB1 mutational status in sporadic DTs. The correlation between sporadic DTs and miRNA expression showed that miR-21-3p increased in mutated versus wild-type DTs, while miR-197-3p was decreased. The mRNA expression of Tetraspanin3 and Serpin family A member 3, as miR-21-3p targets, and L1 Cell Adhesion Molecule, as miR-197-3p target, was also evaluate. CTNNB1 mutations associated to miRNA dysregulation could affect the genesis and the progression of this disease and help histological diagnosis of sporadic DTs.

Characterization of global human microRNA expression patterns across fetal plasma, placenta, and maternal plasma

Characterization of global human microRNA expression patterns across fetal plasma, placenta, and maternal plasma

Abstract 966: Circulating exosomal miRNAs as potential biomarker in liposarcoma

Abstract Liposarcoma (LPS) is the most common soft tissue sarcoma histological subtype. Despite the development of combined modality treatments in recent years, a significant proportion of patients respond poorly to chemotherapy, leading to local recurrence or distant metastasis. Thus, early detection of recurrent or metastatic disease or early decision making could improve patient prognosis. However, there are no useful biomarkers for these purposes. Thus, the discovery of novel biomarkers to detect tumors, predict their drug sensitivity, and monitor their progression is one of the most important challenges that must be overcome. miRNAs are short (approximately 22 nucleotides in length) non-coding RNAs (ncRNAs) that regulate gene expression by binding to specific mRNA targets and promoting their degradation and/or translational inhibition; since their discovery in body fluids (secreted in cell-borne membrane vesicles or associated with macromolecular complexes), circulating blood-borne microRNAs are being investigated as potent minimally invasive biomarkers of several diseases including tumors. In this study, our goal is to identify potential biomarkers in the peripheral blood of LPS patients that could be useful for LPS diagnostic, prognostic and therapeutic purposes. To determine the miRNA expression profiles of LPS in peripheral blood (PB) and in peripheral blood exosomes (PBX), a series of 16 human LPS patient specimens and 8 healthy controls was analyzed on a miRNA microarray platform capable of detecting 800 human miRNAs (nCounter Human microRNA expression Assay Nanostring v3). Validation of the array was performed by qRT-PCR on a new set of samples (10 human LPS and 9 healthy controls). To identify differentially expressed miRNAs, we compared miRNAs expression profiles from PBX of 15 patients to 8 healthy samples. A total of 26 miRNAs were significantly upregulated and 3 miRNAs were significantly down regulated (p&amp;lt;0.004). When we compared miRNA expression profiles from PB to healthy controls, fewer miRNAs were consistently deregulated (18 miRNAs were upregulated and 3 miRNAs were downregulated, p&amp;lt;0.02). Moreover the miRNAs found to be consistently deregulated in PB were all also significantly deregulated in the PBX. Validation of the miRNA panel in an independent cohort of LPS patients confirmed this signature. Our study has identified a specific miRNA signature in circulating exosomes that may have a novel role in the diagnosis, prognosis and or treatment of LPS patients in a clinical setting. Citation Format: Lucia Casadei, Chad Creighton, Kara Batte, Dina Chelouche-Lev, Carlo M. Croce, Raphael E. Pollock. Circulating exosomal miRNAs as potential biomarker in liposarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 966.

Neuro-Epigenetic Indications of Acute Stress Response in Humans: The Case of MicroRNA-29c

Stress research has progressively become more integrative in nature, seeking to unfold crucial relations between the different phenotypic levels of stress manifestations. This study sought to unravel stress-induced variations in expression of human microRNAs sampled in peripheral blood mononuclear cells and further assess their relationship with neuronal and psychological indices.We obtained blood samples from 49 healthy male participants before and three hours after performing a social stress task, while undergoing functional magnetic resonance imaging (fMRI). A seed-based functional connectivity (FC) analysis was conducted for the ventro-medial prefrontal cortex (vmPFC), a key area of stress regulation. Out of hundreds of microRNAs, a specific increase was identified in microRNA-29c (miR-29c) expression, corresponding with both the experience of sustained stress via self-reports, and alterations in vmPFC functional connectivity. Explicitly, miR-29c expression levels corresponded with both increased connectivity of the vmPFC with the anterior insula (aIns), and decreased connectivity of the vmPFC with the left dorso-lateral prefrontal cortex (dlPFC). Our findings further revealed that miR-29c mediates an indirect path linking enhanced vmPFC-aIns connectivity during stress with subsequent experiences of sustained stress. The correlative patterns of miR-29c expression and vmPFC FC, along with the mediating effects on subjective stress sustainment and the presumed localization of miR-29c in astrocytes, together point to an intriguing assumption; miR-29c may serve as a biomarker in the blood for stress-induced functional neural alterations reflecting regulatory processes. Such a multi-level model may hold the key for future personalized intervention in stress psychopathology.

Expression of BKV and JCV encoded microRNA in human cerebrospinal fluid, plasma and urine

BackgroundBK and JC polyomaviruses encode microRNAs which may facilitate the establishment of persistent infection. MicroRNAs contribute to disease pathogenesis, and may provide useful tools in laboratory diagnostics and patient management. ObjectivesIn this pilot work we studied whether viral and cellular microRNAs can be extracted and detected from body fluids to provide added value in a diagnostic laboratory. Study designAltogether 120 human plasma, urine, and cerebrospinal fluid samples from individuals diagnosed with, or suspected of, a severe polyomavirus associated disease, were included in the study. The samples were spiked with unrelated synthetic microRNA to control for sample quality and inhibition. BKV specific bkv-miR-B1-5p, JCV specific jcv-miR-J1-5p, and bkv-miR-B1-3p/jcv-miR-J1-3p, sharing identical sequences between the two viruses, were amplified from human samples using specific TaqMan assays. Expression of 84 circulating human microRNAs was studied in four selected plasma samples in microarray. Resultsjcv-miR-J1-5p and bkv-miR-B1-3p/jcv-miR-J1-3p were frequently amplified from human plasma, urine, and cerebrospinal fluid samples. bkv-miR-B1-5p was amplified from one-third of the samples, which often contained high viral DNA loads. A microarray screen of human microRNAs in plasma samples suggested regulation of several human microRNA expression in BKV positive vs negative samples. ConclusionsViral and cellular microRNAs can be processed and detected from human body fluids. They may prove useful in the diagnosis and management of severe polyomavirus associated diseases, calling for further clinical evaluation.

Possible germ cell-Sertoli cell interactions are critical for establishing appropriate expression levels for the Sertoli cell-specific MicroRNA, miR-202-5p, in human testis.

BackgroundTo examine human microRNA expression in fertile men and subsequently to compare expression patterns of miRNAs in fertile and infertile men, specifically men with Sertoli Cell Only (SCO) histopathology.MethodsTesticular tissues from men with azoospermia and SCO, as well as those of men with normal spermatogenesis, were analyzed. MicroRNA was isolated using the miRCURY™ RNA Purification Kit. A miRCURY LNA™ Universal RT system was used for detection of microRNA by quantitative real-time PCR. MicroRNA localization was performed by in situ hybridizations (ISH) on formalin-fixed paraffin embedded (FFPE) tissue utilizing miRCURY LNA™ microRNA ISH technology. Statistical analysis was performed by GenEx V5.0.ResultsMicroRNA expression was determined for 13 normal fertile men and 5 men with the confirmed diagnosis of diffuse SCO. MiR-202-5p expression was reduced by 17-fold (P < 0.00001) in tissue from SCO men compared to normal. MiR-34c-5p was reduced by 346-fold (P < 0.00001), miR-10b was reduced 18-fold (P < 0.00001), miR-191 was reduced 20-fold (P = 0.001) and miR-126 was reduced 40-fold (P < 0.00001)) in tissues from SCO compared to normal fertile men. Using ISH, miR-202-5p was localized to Sertoli cells of men with normal spermatogenesis, but not in the Sertoli cells of men with SCO.ConclusionNumber of miRNAs are differentially expressed in normal fertile men compared to men with SCO. MicroRNA-202-5p is localized to Sertoli cells and its expression dramatically differs between fertile men and men whose germ cells are depleted, suggesting a novel interaction for regulating microRNA expression between the somatic and germ cell components of the seminiferous epithelium.

Abstract 540: Characterization of miR-9 expression and activation in canine osteosarcoma

Abstract Introduction: Osteosarcoma (OSA) is the most common malignant bone tumor in children and dogs. Despite aggressive treatment, no improvement in survival times has been achieved in the past 15 years with 40% of children and 90% of dogs still dying from disease. MicroRNAs (miRs) are non-protein coding RNAs that have been implicated as having a fundamental role in cancer. We analyzed primary canine OSA tumors and canine osteoblasts for miR expression and found miR-9 to be highly expressed in OSA tumors and cell lines as compared to normal osteoblasts. We hypothesize that miR-9 overexpression generates a pattern of gene dysregulation that contributes to the pathogenesis of OSA. Methodology: Canine bone marrow derived stem cells were differentiated in vitro into osteoblasts and expression of bone-specific markers (ALP, OP, BMP-2) was detected by immunocytochemistry or RT-PCR. MiR expression profiling was performed on 48 primary canine OSA tumors and 3 osteoblast cultures using the NanoString nCounter human microRNA Expression Assay. Taqman miRNA assays were used to measure miR-9 expression in primary canine OSA tumors, OSA cell lines, primary osteoblast cultures, and commercially available osteoblast cell lines. Canine OSA16 and osteoblast cell lines were transduced with lentiviral pre-miR-9 or empty control vector constructs and cells were evaluated for differences in proliferative capacity, apoptosis, and the ability to migrate through Matrigel. Results and Conclusions: We identified 84 miRs (p ≤ 0.01) differentially expressed in primary canine OSA tumors compared to canine osteoblasts. MiR-9 was highly expressed in primary OSA tumors and cell lines as compared to normal osteoblast cell lines or primary cultures. Canine OSA16 and normal osteoblast cell lines, which express low levels of miR-9 were transduced with miR-9 lentiviral constructs resulting in high levels of miR-9 expression. Overexpression of miR-9 did not effect cell proliferation or apoptosis, but enhanced invasion through matrigel in both malignant OSA cells and normal osteoblasts. Our findings suggest that miR-9 may play an important role in regulating OSA cell invasion and suggest that overexpression of miR-9 in vivo may promote metastasis in canine OSA. Transcriptional and proteomic profiling of osteoblasts overexpressing miR-9 is underway to define key differences in mRNA and protein expression and phosphorylation that may contribute to the metastatic phenotype in canine OSA. This work will provide significant new data regarding the impact of miR-9 on molecular pathways in OSA, thereby laying the foundation for the development and testing of novel therapeutics in both children and dogs affected by this disease. Citation Format: Joelle M. Fenger, Jason I. Couto, Misty D. Bear, Stefano Volinia, Jaime F. Modiano, Matthew Breen, Cheryl A. London, William C. Kisseberth. Characterization of miR-9 expression and activation in canine osteosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 540. doi:10.1158/1538-7445.AM2014-540

Differential microRNA expression signatures and cell type-specific association with Taxol resistance in ovarian cancer cells

Paclitaxel (Taxol) resistance remains a major obstacle for the successful treatment of ovarian cancer. MicroRNAs (miRNAs) have oncogenic and tumor suppressor activity and are associated with poor prognosis phenotypes. miRNA screenings for this drug resistance are needed to estimate the prognosis of the disease and find better drug targets. miRNAs that were differentially expressed in Taxol-resistant ovarian cancer cells, compared with Taxol-sensitive cells, were screened by Illumina Human MicroRNA Expression BeadChips. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to identify target genes of selected miRNAs. Kaplan–Meier survival analysis was applied to identify dysregulated miRNAs in ovarian cancer patients using data from The Cancer Genome Atlas. A total of 82 miRNAs were identified in ovarian carcinoma cells compared to normal ovarian cells. miR-141, miR-106a, miR-200c, miR-96, and miR-378 were overexpressed, and miR-411, miR-432, miR-494, miR-409-3p, and miR-655 were underexpressed in ovarian cancer cells. Seventeen miRNAs were overexpressed in Taxol-resistant cells, including miR-663, miR-622, and HS_188. Underexpressed miRNAs in Taxol-sensitive cells included miR-497, miR-187, miR-195, and miR-107. We further showed miR-663 and miR-622 as significant prognosis markers of the chemo-resistant patient group. In particular, the downregulation of the two miRNAs was associated with better survival, perhaps increasing the sensitivity of cancer cells to Taxol. In the chemo-sensitive patient group, only miR-647 could be a prognosis marker. These miRNAs inhibit several interacting genes of p53 networks, especially in TUOS-3 and TUOS-4, and showed cell line-specific inhibition effects. Taken together, the data indicate that the three miRNAs are closely associated with Taxol resistance and potentially better prognosis factors. Our results suggest that these miRNAs were successfully and reliably identified and would be used in the development of miRNA therapies in treating ovarian cancer.

Abstract 184: Breed-associated differential microRNA expression in canine osteosarcoma

Abstract Introduction: MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. miRNAs are dysregulated in cancer, suggesting they play a role in tumorigenesis. Osteosarcoma (OSA) is the most common bone tumor in dogs, however, little is known regarding mechanisms underlying malignant transformation in these tumors. Breeds such as Rottweilers and Greyhounds are at higher risk for developing OSA, suggesting that heritable factors play a role in this disease. We hypothesize that dysregulation of miRNAs in canine OSA is associated with specific breeds. Methods: RNA was isolated by the Trizol (Invitrogen) method from a panel of seven normal canine tissues and 48 primary canine OSA tumors from Greyhound, Golden Retriever, Rottweiler, and mixed breed dogs. miRNA expression was analyzed using the NanoString nCounter human microRNA Expression Assay, interrogating the expression of 752 human miRNAs; 168 of whose mature sequences are 100% conserved between human and dog (Sanger miRBase V15). Samples were hybridized to reporter CodeSets, processed on the nCounter prep station, and scanned with the nCounter Digital Analyzer. Nanominer software was used to perform data normalization. Real time PCR was performed using Applied Biosystems Taqman miRNA assays. Normalization was performed with U6 snRNA and miRNA expression was calculated utilizing the comparative Ct method. P-values of &amp;lt;0.05 were considered statistically significant. For reverse-transcription PCR reactions, cDNA was prepared using Superscript III (Invitrogen) and PCR was performed using ThermoPrime Taq Polymerase (ThermoScientific). Results: miRNA profiling of normal canine tissues revealed tissue-specific miRNA expression signatures. Real time PCR validation of tissue-specific miRNAs validated the use of the NanoString nCounter Assay as a platform for evaluating miRNA expression in canine tissues. Supervised hierarchical cluster analysis revealed distinct breed-associated miRNA expression signatures in canine OSA. 189 miRNAs were differentially expressed in Greyhound, Rottweiler, Golden Retriever, and mixed breed tumors (p&amp;lt;0.01). In an expanded cohort of Greyhound and Rottweiler tumors, real time PCR demonstrated that miR-494 is highly expressed in Rottweiler OSA as compared to Greyhound OSA (p&amp;lt;0.05) or normal canine osteoblasts from various dog breeds, including one Rottweiler. Studies are underway to assess the biological consequences of miR-494 overexpression in normal canine osteoblasts and OSA cell lines. Small RNA sequencing of these OSA samples using the Applied Biosystems SOLiD 4 sequencing platform is being performed to further define breed-associated miRNA signatures in canine OSA. Conclusions: These data reveal significant correlations between breed and miRNA expression in canine OSA, suggesting breed-associated patterns of miRNA dysregulation may play a role in the pathogenesis of OSA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 184. doi:1538-7445.AM2012-184

Functional microRNA screening using a comprehensive lentiviral human microRNA expression library

BackgroundMicroRNAs (miRNAs) are a class of small regulatory RNAs that target sequences in messenger RNAs (mRNAs) to inhibit their protein output. Dissecting the complexities of miRNA function continues to prove challenging as miRNAs are predicted to have thousands of targets, and mRNAs can be targeted by dozens of miRNAs.ResultsTo systematically address biological function of miRNAs, we constructed and validated a lentiviral miRNA expression library containing 660 currently annotated and 422 candidate human miRNA precursors. The miRNAs are expressed from their native genomic backbone, ensuring physiological processing. The arrayed layout of the library renders it ideal for high-throughput screens, but also allows pooled screening and hit picking. We demonstrate its functionality in both short- and long-term assays, and are able to corroborate previously described results of well-studied miRNAs.ConclusionsWith the miRNA expression library we provide a versatile tool for the systematic elucidation of miRNA function.