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Abstract: Tumor viruses are a class of pathogens with well established roles in the development of malignant diseases. Numerous bodies of work have highlighted miRNAs (microRNAs) as critical regulators of tumor pathways and it is clear that the dysregulation of cellular miRNA expression can promote tumor formation. Tumor viruses encode their own miRNAs and/or manipulate the expression of cellular miRNAs to modulate their host cell environment, thereby facilitating their respective infection cycles. The modulation of these miRNA responsive pathways, however, often influences certain signal transduction cascades in ways that favor tumorigenesis. In this review, we discuss the roles of virally-encoded and virally-regulated cellular miRNAs in the respective viral life cycles and in virus associated pathogenesis. [Copyright &y& Elsevier]

The Epstein Barr virus (EBV) contributes to the tumor phenotype through a limited set of primarily non-coding viral RNAs, including 31 mature miRNAs. Here we investigated the impact of EBV miRNAs on remodeling the tumor cell transcriptome. Strikingly, EBV miRNAs displayed exceptionally abundant expression in primary EBV-associated Burkitt's Lymphomas (BLs) and Gastric Carcinomas (GCs). To investigate viral miRNA targeting, we used the high-resolution approach, CLASH in GC and BL cell models. Affinity constant calculations of targeting efficacies for CLASH hits showed that viral miRNAs bind their targets more effectively than their host counterparts, as did Kaposi's sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) miRNAs. Using public BL and GC RNA-seq datasets, we found that high EBV miRNA targeting efficacies translates to enhanced reduction of target expression. Pathway analysis of high efficacy EBV miRNA targets showed enrichment for innate and adaptive immune responses. Inhibition of the immune response by EBV miRNAs was functionally validated in vivo through the finding of inverse correlations between EBV miRNAs and immune cell infiltration and T-cell diversity in BL and GC datasets. Together, this study demonstrates that EBV miRNAs are potent effectors of the tumor transcriptome that play a role in suppressing host immune response. Author summary: Burkitt's Lymphoma and gastric cancer are both associated with EBV, a prolific DNA tumor virus that latently resides in nearly all human beings. Despite mostly restricting viral gene expression to noncoding RNAs, EBV has important influences on the fitness of infected tumor cells. Here, we show that the miRNA class of viral noncoding RNAs are a major viral contributor to remodeling the tumor cell regulatory machinery in patient tumor samples. First, an assessment of miRNA expression in clinical tumor samples showed that EBV miRNAs are expressed at unexpectedly high levels relative to cell miRNAs. Using a highly specific miRNA target identification approach, CLASH, we comprehensively identified both viral and cellular miRNA targets and the relative abundance of each miRNA-mRNA interaction. We also show that viral miRNAs bind to and alter the expression of their mRNA targets more effectively than their cellular miRNA counterparts. Pathway analysis of the most effectively targeted mRNAs revealed enrichment of immune signaling pathways and we show a corresponding inverse correlation between EBV miRNA expression and infiltrating immune cells in EBV positive primary tumors. Altogether, this study shows that EBV miRNAs are key regulators of the tumor cell phenotype and the immune cell microenvironment. [ABSTRACT FROM AUTHOR]

Ovarian carcinoma is the fifth-leading cause of cancer death among women in the United States. Major reasons for this persistent mortality include the poor understanding of the underlying biology and a lack of reliable biomarkers. Previous studies have shown that aberrantly expressed MicroRNAs (miRNAs) are involved in carcinogenesis and tumor progression by post-transcriptionally regulating gene expression. However, the interference of miRNAs in tumorigenesis is quite complicated and far from being fully understood. In this work, by an integrative analysis of mRNA expression, miRNA expression and clinical data published by The Cancer Genome Atlas (TCGA), we studied the modularity and dynamicity of miRNA–mRNA interactions and the prognostic implications in high-grade serous ovarian carcinomas. With the top transcriptional correlations (Bonferroni-adjusted p -value < 0.01) as inputs, we identified five miRNA–mRNA module pairs (MPs), each of which included one positive-connection (correlation) module and one negative-connection (correlation) module. The number of miRNAs or mRNAs in each module varied from 3 to 7 or from 2 to 873. Among the four major negative-connection modules, three fit well with the widely accepted miRNA-mediated post-transcriptional regulation theory. These modules were enriched with the genes relevant to cell cycle and immune response. Moreover, we proposed two novel algorithms to reveal the group or sample specific dynamic regulations between these two RNA classes. The obtained miRNA–mRNA dynamic network contains 3350 interactions captured across different cancer progression stages or tumor grades. We found that those dynamic interactions tended to concentrate on a few miRNAs (e.g. miRNA-936), and were more likely present on the miRNA–mRNA pairs outside the discovered modules. In addition, we also pinpointed a robust prognostic signature consisting of 56 modular protein-coding genes, whose co-expression patterns were predictive for the survival time of ovarian cancer patients in multiple independent cohorts. [ABSTRACT FROM AUTHOR]

The relationship between Epstein Barr Virus (EBV) and miR-155 is well established. EBV infection induces miR-155 expression, which is expressed at higher levels in EBV latency type III cells compared to EBV latency type I cells. However, the mechanism by which EBV latency genes activate miR-155 expression is still unclear. Here we present data showing that DNA methylation regulates miR-155 expression. We also provide evidence that the AP1 signaling pathway is involved in EBV-mediated miR-155 activation, and that Bay11 influences signaling of the miR-155 promoter AP1 element. Lastly, we show that LMP2A, LMP1 and EBNAs cannot activate miR-155 expression alone, indicating that the regulation of miR-155 by EBV is dependent on more than one EBV gene or cell signaling pathway. We conclude that the regulation of miR-155 in EBV-positive cells occurs through multiple cell signaling processes involving EBV-mediated chromatin remodeling, cell signaling regulation and transcription factor activation. [ABSTRACT FROM AUTHOR]

In the oral epithelium, peripheral stores of Epstein-Barr virus (EBV) are transmitted from infiltrating B cells to epithelial cells. Once the virus is transmitted to epithelial cells, the highly permissive nature of this cell type for lytic replication allows virus amplification and exchange to other hosts. Since the initial transfer of EBV from B cells to epithelial cells requires transitioning of the B-cell to a state that induces virus reactivation, we hypothesized that there might be epithelium-specific signals that allow the infiltrating B cells to sense the appropriate environment to initiate reactivation and begin this exchange process. We previously found that the epithelium-specific miR-200 family of microRNAs promotes EBV lytic replication. Here we show that there are high levels of miR-200 family members in oral and tonsillar epithelia and in saliva. Analysis of cultured oral epithelial cells (OKF6) showed that they actively secrete membrane vesicles (exosomes) that are enriched with miR-200 family members. Coculturing of EBV-positive B cells with OKF6 cells induced viral reactivation. Further, treatment of EBV-positive B cells with OKF6 cell-derived membrane vesicles promoted reactivation. Using a cell system that does not naturally express miR-200 family members, we found that enforced expression of a miR-200 family member produced membrane vesicles that were able to induce the lytic cascade in EBV-positive B cells. We propose that membrane vesicles secreted by oral and tonsillar epithelial cells may serve as a tissue-specific environmental cue that initiates reactivation in B cells, promoting the transfer of virus from peripheral B-cell stores to the oral epithelium to facilitate virus amplification and exchange to other hosts. [ABSTRACT FROM AUTHOR]

Background: The AKT/mammalian target of rapamycin (mTOR) signaling pathway is regulated by 17α-estradiol (E2) signaling and mediates E2-induced proliferation and progesterone receptor (PgR) expression in breast cancer. Methods and results: Here we use deep sequencing analysis of previously published data from The Cancer Genome Atlas to demonstrate that expression of a key component of mTOR signaling, rapamycin-insensitive companion of mTOR (Rictor), positively correlated with an estrogen receptor-α positive (ERα+) breast tumor signature. Through increased microRNA-155 (miR-155) expression in the ERα+ breast cancer cells we demonstrate repression of Rictor enhanced activation of mTOR complex 1 (mTORC1) signaling with both qPCR and western blot. miR-155-mediated mTOR signaling resulted in deregulated ERα signaling both in cultured cells in vitro and in xenografts in vivo in addition to repressed PgR expression and activity. Furthermore we observed that miR-155 enhanced mTORC1 signaling (observed through western blot for increased phosphorylation on mTOR S2448) and induced inhibition of mTORC2 signaling (evident through repressed Rictor and tuberous sclerosis 1 (TSC1) gene expression). mTORC1 induced deregulation of E2 signaling was confirmed using qPCR and the mTORC1-specific inhibitor RAD001. Co-treatment of MCF7 breast cancer cells stably overexpressing miR-155 with RAD001 and E2 restored E2-induced PgR gene expression. RAD001 treatment of SCID/CB17 mice inhibited E2-induced tumorigenesis of the MCF7 miR-155 overexpressing cell line. Finally we demonstrated a strong positive correlation between Rictor and PgR expression and a negative correlation with Raptor expression in Luminal B breast cancer samples, a breast cancer histological subtype known for having an altered ERα-signaling pathway. Conclusions: miRNA mediated alterations in mTOR and ERα signaling establishes a new mechanism for altered estrogen responses independent of growth factor stimulation. [ABSTRACT FROM AUTHOR]

Abstract: Accumulating evidence indicates that elevated S100P promotes the pathogenesis of cancers, including colon cancer. S100P exerts its effects by binding to and activating the Receptor for Advance Glycation End-products (RAGE). The effects of up-regulated S100P/RAGE signaling on cell functions are well documented. Despite these observations, little is known about the downstream targets of S100P/RAGE signaling. In the present study, we demonstrated for the first time that activation of RAGE by S100P regulates oncogenic microRNA-155 (miR-155) expression through Activator Protein-1 (AP-1) stimulation in colon cancer cells. Ectopic S100P up-regulated miR-155 levels in human colon cancer cells. Conversely, knockdown of S100P resulted in a decrease in miR-155 levels. Exogenous S100P induced miR-155 expression, but blockage of the RAGE with anti-RAGE antibody suppressed the induction of miR-155 by exogenous S100P. Attenuation of AP-1 activation through pharmacological inhibition of MEK activation or genetic inhibition of c-Jun activation using dominant negative c-Jun (TAM67) suppressed miR-155 induction by exogenous S100P. Also, S100P treatment stimulated the enrichment of c-Fos, an AP-1 family member, at the miR-155 host gene promoter site. Finally, a functional study demonstrated that miR-155 knockdown decreases colon cancer cell growth, motility, and invasion. Altogether, these data demonstrate that the expression of miR-155 is regulated by S100P and is dependent on RAGE activation and stimulation of AP-1. [Copyright &y& Elsevier]

The count of the nucleotides in a cloned, short genomic sequence has become an important criterion to annotate such a sequence as a miRNA molecule. While the majority of human mature miRNA sequences consist of 22 nucleotides, there exists discrepancy in the characteristic lengths of the miRNA sequences. There is also a lack of systematic studies on such length distribution and on the biological factors that are related to or may affect this length. In this paper, we intend to fill this gap by investigating the sequence structure of human miRNA molecules using statistics tools. We demonstrate that the traditional discrete probability distributions do not model the length distribution of the human mature miRNAs well, and we obtain the statistical distribution model with a decent fit. We observe that the four nucleotide bases in a miRNA sequence are not randomly distributed, implying that possible structural patterns such as dinucleotide (trinucleotide or higher order) may exist. Furthermore, we study the relationships of this length distribution to multiple important factors such as evolutionary conservation, tumorigenesis, the length of precursor loop structures, and the number of predicted targets. The association between the miRNA sequence length and the distributions of target site counts in corresponding predicted genes is also presented. This study results in several novel findings worthy of further investigation that include: (1) rapid evolution introduces variation to the miRNA sequence length distribution; (2) miRNAs with extreme sequence lengths are unlikely to be cancer-related; and (3) the miRNA sequence length is positively correlated to the precursor length and the number of predicted target genes. [ABSTRACT FROM AUTHOR]

Abstract: Three-dimensional organotypic culture using reconstituted basement membrane matrix (rBM 3-D) is an invaluable tool to characterize morphogenesis of epithelial cells and to elucidate the tumor-modulating actions of extracellular matrix. microRNAs (miRNA) are a novel class of tumor modulating genes. A substantial amount of investigation of miRNAs in cancer is carried out using monolayer 2-D culture on plastic substratum, which lacks a consideration of the matrix-mediated regulation of miRNAs. In the current study we compared the expression of miRNAs in rBM 3-D and 2-D cultures of two lung adenocarcinoma cell lines. Our findings revealed a profound difference in miRNA profiles between 2-D and rBM 3-D cultures of lung adenocarcinoma cells. The rBM 3-D culture-specific miRNA profile was highlighted with higher expression of the tumor suppressive miRNAs (i.e., miR-200 family) and lower expression of the oncogenic miRNAs (i.e., miR-17–92 cluster and miR-21) than that of 2-D culture. Moreover, the expression pattern of miR-17, miR-21, and miR-200a in rBM 3-D culture correlated with the expression of their targets and acinar morphogenesis, a differentiation behavior of lung epithelial cells in rBM 3-D culture. Over-expression of miR-21 suppressed its target PTEN and disrupted acinar morphogenesis. In summary, we provide the first miRNA profile of lung adenocarcinoma cells in rBM 3-D culture with respect to acinar morphogenesis. These results indicate that rBM 3-D culture is essential to a comprehensive understanding of the miRNA biology in lung epithelial cells pertinent to lung adenocarcinoma. [Copyright &y& Elsevier]

Recent studies have shown the contribution of miRNAs to cancer pathogenesis. Prostate cancer is the most commonly diagnosed cancer in men. Unlike other major types of cancer, no single gene has been identified as being mutated in the majority of prostate tumors. This implies that the expression profiling of genes, including the non-coding miRNAs, may substantially vary across individual cases of this cancer. The within-class variability makes it possible to reconstruct or infer disease-specific miRNA-mRNA correlation and regulatory modular networks using high-dimensional microarray data of prostate tumor samples. Furthermore, since miRNAs and tumor suppressor genes are usually tissue specific, miRNA-mRNA modules could potentially differ between primary prostate cancer (PPC) and metastatic prostate cancer (MPC). We herein performed an in silico analysis to explore the miRNA-mRNA correlation network modules in the two tumor subtypes. Our analysis identified 5 miRNA-mRNA module pairs (MPs) for PPC and MPC, respectively. Each MP includes one positiveconnection (correlation) module and one negative-connection (correlation) module. The number of miRNAs or mRNAs (genes) in each module varies from 2 to 8 or from 6 to 622. The modules discovered for PPC are more informative than those for MPC in terms of the implicated biological insights. In particular, one negative-connection module in PPC fits well with the popularly recognized miRNA-mediated post-transcriptional regulation theory. That is, the 3'UTR sequences of the involved mRNAs (∼620) are enriched with the target site motifs of the 7 modular miRNAs, has-miR-106b, -191, -19b, -92a, - 92b, -93, and -141. About 330 GO terms and KEGG pathways, including TGF-beta signaling pathway that maintains tissue homeostasis and plays a crucial role in the suppression of the proliferation of cancer cells, are over-represented (adj.p<0.05) in the modular gene list. These computationally identified modules provide remarkable biological evidence for the interference of miRNAs in the development of prostate cancers and warrant additional follow-up in independent laboratory studies. [ABSTRACT FROM AUTHOR]

Abstract: The Epstein–Barr virus (EBV) is associated with lymphoid and epithelial cancers. Initial EBV infection alters lymphocyte gene expression, inducing cellular proliferation and differentiation as the virus transitions through consecutive latency transcription programs. Cellular microRNAs (miRNAs) are important regulators of signaling pathways and are implicated in carcinogenesis. The extent to which EBV exploits cellular miRNAs is unknown. Using micro-array analysis and quantitative PCR, we demonstrate differential expression of cellular miRNAs in type III versus type I EBV latency including elevated expression of miR-21, miR-23a, miR-24, miR-27a, miR-34a, miR-146a and b, and miR-155. In contrast, miR-28 expression was found to be lower in type III latency. The EBV-mediated regulation of cellular miRNAs may contribute to EBV signaling and associated cancers. [Copyright &y& Elsevier]