Identifying microRNA/mRNA dysregulations in ovarian cancer

Abstract Background MicroRNAs are a class of noncoding RNA molecules that co-regulate the expression of multiple genes via mRNA transcript degradation or translation inhibition. Since they often target entire pathways, they may be better drug targets than genes or proteins. MicroRNAs are known to be dysregulated in many tumours and associated with aggressive or poor prognosis phenotypes. Since they regulate mRNA in a tissue specific manner, their functional mRNA targets are poorly understood. In previous work, we developed a method to identify direct mRNA targets of microRNA using patient matched microRNA/mRNA expression data using an anti-correlation signature. This method, applied to clear cell Renal Cell Carcinoma (ccRCC), revealed many new regulatory pathways compromised in ccRCC. In the present paper, we apply this method to identify dysregulated microRNA/mRNA mechanisms in ovarian cancer using data from The Cancer Genome Atlas (TCGA). Methods TCGA Microarray data was normalized and samples whose class labels (tumour or normal) were ambiguous with respect to consensus ensemble K-Means clustering were removed. Significantly anti-correlated and correlated genes/microRNA differentially expressed between tumour and normal samples were identified. TargetScan was used to identify gene targets of microRNA. Results We identified novel microRNA/mRNA mechanisms in ovarian cancer. For example, the expression level of RAD51AP1 was found to be strongly anti-correlated with the expression of hsa-miR-140-3p, which was significantly down-regulated in the tumour samples. The anti-correlation signature was present separately in the tumour and normal samples, suggesting a direct causal dysregulation of RAD51AP1 by hsa-miR-140-3p in the ovary. Other pairs of potentially biological relevance include: hsa-miR-145/E2F3, hsa-miR-139-5p/TOP2A, and hsa-miR-133a/GCLC. We also identified sets of positively correlated microRNA/mRNA pairs that are most likely result from indirect regulatory mechanisms. Conclusions Our findings identify novel microRNA/mRNA relationships that can be verified experimentally. We identify both generic microRNA/mRNA regulation mechanisms in the ovary as well as specific microRNA/mRNA controls which are turned on or off in ovarian tumours. Our results suggest that the disease process uses specific mechanisms which may be significant for their utility as early detection biomarkers or in the development of microRNA therapies in treating ovarian cancers. The positively correlated microRNA/mRNA pairs suggest the existence of novel regulatory mechanisms that proceed via intermediate states (indirect regulation) in ovarian tumorigenesis.</p

Patterns of microRNA Expression in Non-Human Primate Cells Correlate with Neoplastic Development In Vitro

MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate gene expression post-transcriptionally. They play a critical role in developmental and physiological processes and have been implicated in the pathogenesis of several diseases including cancer. To identify miRNA signatures associated with different stages of neoplastic development, we examined the expression profile of 776 primate miRNAs in VERO cells (a neoplastically transformed cell line being used for the manufacture of viral vaccines), progenitor primary African green monkey kidney (pAGMK) cells, and VERO cell derivatives: spontaneously immortalized, non-tumorigenic, low-passage VERO cells (10-87 LP); tumorigenic, high-passage VERO cells (10-87 HP); and a cell line (10-87 T) derived from a 10-87 HP cell tumor xenograft in athymic nude mice. When compared with pAGMK cells, the majority of miRNAs were expressed at lower levels in 10-87 LP, 10-87 HP, and 10-87 T cells. We identified 10 up-regulated miRNAs whose level of expression correlated with VERO cell evolution from a non-tumorigenic phenotype to a tumorigenic phenotype. The overexpression of miR-376a and the polycistronic cluster of miR-376a, miR-376b and miR-376c conferred phenotypic changes to the non-tumorigenic 10-87 LP cells that mimic the tumorigenic 10-87 HP cells. Thirty percent of miRNAs that were components of the identified miRNAs in our spontaneously transformed AGMK cell model are also dysregulated in a variety of human tumors. These results may prove to be relevant to the biology of neoplastic development. In addition, one or more of these miRNAs could be biomarkers for the expression of a tumorigenic phenotype

Motif co-regulation and co-operativity are common mechanisms in transcriptional, post-transcriptional and post-translational regulation

A substantial portion of the regulatory interactions in the higher eukaryotic cell are mediated by simple sequence motifs in the regulatory segments of genes and (pre-)mRNAs, and in the intrinsically disordered regions of proteins. Although these regulatory modules are physicochemically distinct, they share an evolutionary plasticity that has facilitated a rapid growth of their use and resulted in their ubiquity in complex organisms. The ease of motif acquisition simplifies access to basal housekeeping functions, facilitates the co-regulation of multiple biomolecules allowing them to respond in a coordinated manner to changes in the cell state, and supports the integration of multiple signals for combinatorial decision-making. Consequently, motifs are indispensable for temporal, spatial, conditional and basal regulation at the transcriptional, post-transcriptional and post-translational level. In this review, we highlight that many of the key regulatory pathways of the cell are recruited by motifs and that the ease of motif acquisition has resulted in large networks of co-regulated biomolecules. We discuss how co-operativity allows simple static motifs to perform the conditional regulation that underlies decision-making in higher eukaryotic biological systems. We observe that each gene and its products have a unique set of DNA, RNA or protein motifs that encode a regulatory program to define the logical circuitry that guides the life cycle of these biomolecules, from transcription to degradation. Finally, we contrast the regulatory properties of protein motifs and the regulatory elements of DNA and (pre-)mRNAs, advocating that co-regulation, co-operativity, and motif-driven regulatory programs are common mechanisms that emerge from the use of simple, evolutionarily plastic regulatory modules

Malaria transmission blocking activity of sesquiterpene lactones from Vernonia amygdalina

Background: Most of the currently available anti-malarial drugs act on asexual stages of the Plasmodium parasite and have limited impact on the sexual stages to block transmission. Search for drugs active against transmissible stages imperative for the development of transmission blocking interventions. This study aimed to assess whether Vernonia amygdalina, a plant used traditionally to treat malaria fever, contains secondary metabolites interfering with the development of early sporogonic stages (ESS): gamete, zygote formation and/or ookinete maturation. Methods: Plasmodium berghei CTRPp.GFP was used to determine in vitro activity of fractions and isolated molecules against ESS. Gametocytemic blood from BALB/c mice was incubated in microplates with the test substances. Zygote and ookinete formation was scored after 40h of incubation under the fluorescent microscope (400x). The fractions, found active on P. berghei, were then examined on P. falciparum field isolates. Gametocytemic blood from volunteers was supplemented with fractions (at 100 ppm) and membrane fed to Anopheles coluzzii mosquitoes. A week later, mosquito midguts were dissected and examined for oocysts. Results: Organic fractions from the methanol extract of V. amygdalina leaves proved to be strongly active against ESS of P. berghei. Fraction 11 (eluted with n-hexane:ethylacetate 1:1 and 7:13) and fraction 13 (eluted with ethylacetate) suppressed ESS development by 98- 100% at a concentration of 50 ppm . In the experiments with P. falciparum field isolates, control mosquitoes displayed an oocyst prevalence ranging from 30 to 50%, whereas the oocyst prevalence in mosquitoes membrane fed with gametocytemic blood treated with fraction 11 and 13 at 100 ppm ranged from 0 to 32% and 0 to 8%, respectively. The oocyst density among oocyst-positive mosquitoes varied from 3.5 to 4.2 per mosquito in controls, compare to 1.8 to 2.4 and 1.3 to 1.8 in fraction 11 and 13, respectively. Subsequent chemical analysis revealed sesquiterpenes vernolide and vernodalol to be the major components of fraction 11 and 13, respectively. The isolated molecules confirmed their effects on P. berghei ESS in vitro, with vernodalol displaying a relatively stronger inhibitory activity than vernolide on the transmissible stages. Conclusion: Vernolide and vernodalol rich fractions from V. amygdalina leaves hold promise for the development of medicines impacting on the transmissible stages of the malaria parasites