Artificial neural network techniques to investigate potential interactions between biomarkers

High-throughput technologies in biomedical sciences, including gene microarrays, supposed to revolutionise the post-genomic era, have barely met the great expectations they inspired to the biomedical community at first. Current efforts are still focused toward improving the technology, its reproducibility and accuracy. In the meantime, computational techniques for the analysis of the data from these technologies have achieved great progresses and show encouraging results. New approaches have been developed to extract relevant information out from these results. However, important work needs to be further conducted in order to extract even more meaningful and relevant information. These techniques offer great possibilities to explore the overall dynamic held within a living organism. The potential information contained in their output can reveal important leads at deciphering the interconnection, interaction or regulation influences that can exist between several molecules. In front of an increasing interest of the scientific community toward the exploration of these dynamics, some groups have started to develop solutions based on different technologies to extract these information related to interactions. Here we present an Artificial Neural Network-based methodology for the study of interactions in gene transcriptomic data. This will be applied and validated in a breast cancer context

Computational prediction and experimental validation of evolutionarily conserved microRNA target genes in bilaterian animals

<p>Abstract</p> <p>Background</p> <p>In many eukaryotes, microRNAs (miRNAs) bind to complementary sites in the 3'-untranslated regions (3'-UTRs) of target messenger RNAs (mRNAs) and regulate their expression at the stage of translation. Recent studies have revealed that many miRNAs are evolutionarily conserved; however, the evolution of their target genes has yet to be systematically characterized. We sought to elucidate a set of conserved miRNA/target-gene pairs and to analyse the mechanism underlying miRNA-mediated gene regulation in the early stage of bilaterian evolution.</p> <p>Results</p> <p>Initially, we extracted five evolutionarily conserved miRNAs (<it>let-7</it>, <it>miR-1</it>, <it>miR-124</it>, <it>miR-125/lin-4</it>, and <it>miR-34</it>) among five diverse bilaterian animals. Subsequently, we designed a procedure to predict evolutionarily conserved miRNA/target-gene pairs by introducing orthologous gene information. As a result, we extracted 31 orthologous miRNA/target-gene pairs that were conserved among at least four diverse bilaterian animals; the prediction set showed prominent enrichment of orthologous miRNA/target-gene pairs that were verified experimentally. Approximately 84% of the target genes were regulated by three miRNAs (<it>let-7, miR-1</it>, and <it>miR-124</it>) and their function was classified mainly into the following categories: development, muscle formation, cell adhesion, and gene regulation. We used a reporter gene assay to experimentally verify the downregulation of six candidate pairs (out of six tested pairs) in HeLa cells.</p> <p>Conclusions</p> <p>The application of our new method enables the identification of 31 miRNA/target-gene pairs that were expected to have been regulated from the era of the common bilaterian ancestor. The downregulation of all six candidate pairs suggests that orthologous information contributed to the elucidation of the primordial set of genes that has been regulated by miRNAs; it was also an efficient tool for the elimination of false positives from the predicted candidates. In conclusion, our study identified potentially important miRNA-target pairs that were evolutionarily conserved throughout diverse bilaterian animals and that may provide new insights into early-stage miRNA functions.</p

Computational design of orthogonal microRNAs for synthetic biology

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.Upcoming applications of synthetic biology will require access to a wide array of robust genetic components (parts). The logic of a genetic system is encoded with regulatory elements such as pairs of transcription factors:promoters, miRNAs:target sites, or ribozymes:aptamers among others. Due to a relatively simple form and mode of operation of miRNAs, it is possible to design their synthetic variants. Out of all possible miRNA sequences the ones chosen should perform efficiently and should avoid cross-talk with both the host system circuits and within the imported synthetic ones. In this work, a computational method involving a series of heuristics is developed that can be used to design ensembles of such sequences depending on the host transcriptome. As an example, an ensemble of eight such miRNA sequences is produced using this method for use in a human host. Those have then been validated experimentally against the above-mentioned requirements by transfection into HEK 293 cells and flow cytometry measurements of fluorescent markers. The produced sequences are available for use from pENTR vectors of the Gateway cloning system. The required computations were facilitated by a modern cluster computing system—Kaichu—especially developed for this project, but fit for general purpose use and available under an open-source license.EPSR

ABCB5 and the regulation of p16INK4a by non-coding RNA

p16INK4a (p16) traps the cell at the restriction point of the cell cycle by binding to cyclin-dependent kinase 4/6 thus preventing the phosphorylation of the retinoblastoma protein (pRB). As p16 accumulates the cell stops dividing and becomes senescent. This study investigates the modulation of p16 function by the putative membrane protein ABCB5 and a group of five putative oncogenic microRNAs (oncomiRs). ABCB5 is a poorly characterised member of the B-subfamily of human ATP Binding Cassette transporters. ABCB5 is reportedly transcribed into four transcripts, one of which could potentially encode a full-length transporter (ABCB5fl) whilst a second could encode a half-transporter (ABCB5β). The other two transcripts (ABCB5α and ABCB5γ) could only encode short polypeptides. Exogenous expression of ABCB5fl and ABCB5β was achieved in HEK293T cells, but the recombinant protein expressed poorly and localised to the endoplasmic reticulum. Point mutations introduced into the ATP catalytic domain failed to improve expression levels suggesting that protein function was not deleterious to the cell. Exogenous expression in HEK293T cells also allowed commercial antibodies purportedly raised against ABCB5 isoforms to be tested. Several were found not to recognise ABCB5 necessitating re-interpretation of published data. However, one antibody recognised both ABCB5fl and ABCB5β, and was subsequently used to evaluate protein expression levels in other cell types.siRNA knockdown of ABCB5 in human mammary epithelial cells (HMECs) caused a concomitant reduction in p16 expression and an increase in cellular proliferation. Differential siRNAs and RT-qPCR analyses demonstrated ABCB5β to be the relevant transcript with respect to the reduction in p16 expression; however, no native ABCB5β protein was detected in HMECs. Together these data lead to the hypothesis that the ABCB5β transcript may act as a long noncoding RNA to regulate p16. Exogenous expression of each of five distinct putative oncomiRs in HMECs was found to increase cellular proliferation and, surprisingly, increase p16 expression. These results mirror a phenotype commonly observed in p16-positive basal-like breast cancer (BLBC), an aggressive form of breast cancer with poor prognosis and few treatment options. Bioinformatic analysis of the predicted target genes for these oncomiRs identified multiple transcriptional regulators of pRB. These predictions, together with the work performed in a cellular model of p16-positive BLBC, suggest that the oncomiRs may cause unrestricted cell proliferation by indirectly reducing transcription of the pRB gene, RB1. In the absence of pRB, p16 expression is induced via a previously reported oncogeneinduced senescence-like positive feedback loop. These data, and previously published observations, suggest that a similar mechanism may explain the basi

Quantification of Placental Dysfunction in Pregnancy Complications

Background The pathogenetic mechanisms behind placental dysfunction-related complications like preeclampsia and intrauterine growth restriction have remained perplexing till now, in part because of lack of well-defined structural and functional molecular characterisation. There is growing evidence that links trophoblast debris and the existence of syncytial nuclear aggregates (SNA) to the pathogenesis of gestational diseases. Characterisation and quantification of structural and functional parameters of placental dysfunction may give researchers a clearer picture of the mechanisms underlying the development of high risk pregnancy. Methods Placental samples were obtained from normal term pregnancies, preterm controls, as well as from pregnancies complicated by preeclampsia (PET), intrauterine growth restriction (IUGR) and PET-IUGR. Formalin-fixed, paraffin-embedded sections were visualised with H&E, stained using immunohistochemistry (IHC) and digitally scanned. Using stereological methodology, volumes of placental SNAs, trophoblasts, villi and capillaries were measured. Three dimensional (3D) volume reconstructions of terminal placental villi with SNAs and fibrinoid degenerations were created. IHC-labelled slides were analysed by image analysis algorithms. Differential expression of placental genes and miRNAs, hypothesised to regulate cell death in placental dysfunction, were quantified using RT-qPCR. BeWo cell lines were carried out for in vitro validation of the effects miRNAs regulating programmed cell death (PCD) using flow cytometry and western blotting. Results Specific morphometric patterns of villous, trophoblasts, SNA and capillary volumes were demonstrated with characteristic higher SNAs and lower capillary volumes in PET placentae with reciprocal patterns in IUGR placentae showing a negative correlation pattern between nuclear aggregates and capillary volumes. Image analysis of immune-labelled slides showed a higher autophagy marker expression in PET and a positive correlation to SNAs as well as a balanced reciprocal expression patterns with apoptosis. Moreover, miR-204 transfected BeWo cells showed a similar balanced reciprocal regulation of autophagy and apoptosis expressions. Conclusion We have demonstrated that applying stereology-based and image analysis on digitised placental sections can be useful in quantifying and dissecting structural and functional patterns in normal and abnormal placental function. 3D reconstruction model are a novel approach towards placental characterisation in normal and complicated pregnancies. The study also showed that miR-204 plays a vital role in the regulation of placental autophagy and apoptosis, critical in the pathophysiology of placental dysfunction

Quantification of Placental Dysfunction in Pregnancy Complications

Background The pathogenetic mechanisms behind placental dysfunction-related complications like preeclampsia and intrauterine growth restriction have remained perplexing till now, in part because of lack of well-defined structural and functional molecular characterisation. There is growing evidence that links trophoblast debris and the existence of syncytial nuclear aggregates (SNA) to the pathogenesis of gestational diseases. Characterisation and quantification of structural and functional parameters of placental dysfunction may give researchers a clearer picture of the mechanisms underlying the development of high risk pregnancy. Methods Placental samples were obtained from normal term pregnancies, preterm controls, as well as from pregnancies complicated by preeclampsia (PET), intrauterine growth restriction (IUGR) and PET-IUGR. Formalin-fixed, paraffin-embedded sections were visualised with H&E, stained using immunohistochemistry (IHC) and digitally scanned. Using stereological methodology, volumes of placental SNAs, trophoblasts, villi and capillaries were measured. Three dimensional (3D) volume reconstructions of terminal placental villi with SNAs and fibrinoid degenerations were created. IHC-labelled slides were analysed by image analysis algorithms. Differential expression of placental genes and miRNAs, hypothesised to regulate cell death in placental dysfunction, were quantified using RT-qPCR. BeWo cell lines were carried out for in vitro validation of the effects miRNAs regulating programmed cell death (PCD) using flow cytometry and western blotting. Results Specific morphometric patterns of villous, trophoblasts, SNA and capillary volumes were demonstrated with characteristic higher SNAs and lower capillary volumes in PET placentae with reciprocal patterns in IUGR placentae showing a negative correlation pattern between nuclear aggregates and capillary volumes. Image analysis of immune-labelled slides showed a higher autophagy marker expression in PET and a positive correlation to SNAs as well as a balanced reciprocal expression patterns with apoptosis. Moreover, miR-204 transfected BeWo cells showed a similar balanced reciprocal regulation of autophagy and apoptosis expressions. Conclusion We have demonstrated that applying stereology-based and image analysis on digitised placental sections can be useful in quantifying and dissecting structural and functional patterns in normal and abnormal placental function. 3D reconstruction model are a novel approach towards placental characterisation in normal and complicated pregnancies. The study also showed that miR-204 plays a vital role in the regulation of placental autophagy and apoptosis, critical in the pathophysiology of placental dysfunction

Characterisation of oestrogen-responsive microRNAs regulating coagulation factors

Oestradiol (E2) regulation of microRNAs (miRNAs) is well characterised in breast cancer, but poorly understood in regulating coagulation. We previously reported that miR-494 directly downregulates Protein S gene (PROS1) expression in E2-treated human liver carcinoma cells (HuH-7) (Tay et al. 2013). Subsequent miRNA array analyses (NanoString nCounter®) identified numerous E2-responsive miRNAs in HuH-7 cells, which may be involved in regulating thrombotic factors. Therefore, this study sought to validate the E2 responsiveness of the candidate miRNAs and investigate their direct effects on coagulation gene targets. Twelve E2-responsive miRNAs were selected for validation and their potential coagulation gene targets predicted using four computational tools. HuH-7 cells were cultured -/+ 10nM E2 for 12h followed by RT-qPCR quantitation of E2-mediated expression of the twelve selected miRNAs and predicted gene targets. The direct interaction between validated miRNAs and their common coagulation gene targets were measured via dual-luciferase reporter assays. In concordance with NanoString nCounter® results, expression of let-7f-5p, miR-26b-5p, miR-128-3p, miR-365a-3p, miR-455-3p and miR-548aa were significantly downregulated following E2 treatment (p<0.05). An associated increase in common predicted targets Tissue Factor (F3) and Factor VIII (F8) mRNA levels was also observed. Furthermore, miR-365a-3p was identified to have a direct binding site on F3-3’UTR. MiR- 365a-3p was down-regulated by E2 and it could directly bind to F3-3’UTR, suggesting downregulation of miR-365a-3p may lead to up-regulation of Tissue Factor to promote thrombosis in high circulating E2 levels during pregnancy or contraceptive pill use

MicroRNA profile and function in kidney ischaemia and reperfusion injury

PhD ThesisIschaemia and reperfusion injury (IRI) in renal allografts is an important contributing factor to chronic allograft dysfunction. MicroRNAs (miRNA) have been shown to play important roles in cellular adaptation to pathological conditions, including IRI. This study aimed to evaluate changes in miRNA profile following IRI, and how the changes in particular miRNAs may influence renal proximal tubular epithelial cell (PTEC) morphology and function, potentially contributing to the development of chronic allograft dysfunction. To achieve this, several objectives were set. These included: (1) isolation and culture of primary human PTECs, (2) miRNA expression profiling following IRI and selection of candidate miRNA, and (3) in vitro and human pathology validation to explore the molecular mechanism of the candidate miRNA. Primary PTECs were isolated from normal renal tissue. These cells showed features of epithelial cells under light microscope and electron microscope. The cells were also characterised using immunofluorescent staining, which showed positive expression of epithelial cell markers, and negative expression for mesenchymal cell markers. MiRNA profiling using NanoString platform was performed on cell lines (HKC-8 and HK-2) and primary PTECs, which were exposed to either hypoxia or free radicals. Results revealed distinct miRNA signature changes following IRI in cells. However, only a small proportion of microRNAs were found to be significantly up/down-regulated in either cell lines or primary cells, which included miR-21, miR-376, miR-190b, miR-34a, miR-210, miR-363, miR-142 and miR-130b. MiR-21 was shown to be up-regulated in all cells following both type of injuries. Online target prediction analysis also showed miR-21 to be involved in pathways relevant to cellular response to IRI and the development of fibrosis. The role of miR-21 was studied in detail. Up-regulation of miR-21 following ischaemia was shown to suppress SMAD7 and facilitate intra-nuclear localisation of SMAD3. In the presence of exogenous TGF-b1 or hypoxia, over-expression of miR-21 in cells led to an increase in SMAD3 activity. Over-expression of miR-21 also led to phenotypic shift in HKC- 8 cells, characterised as a decrease in E-cadherin and an increase in a-SMA and Collagen-1 expression. Human pathology evaluation confirmed high expression of miR-21 in the tubular cells of severely ischaemic kidneys compared to non-ischaemic kidneys. In conclusion, changes in miRNA profile were observed in acute IRI in the kidney. One of the significantly affected miRNAs was miR-21. MiR-21 up-regulation resulted in sensitisation of tubular cells to TGF-b1, which may be essential in cellular repair processes, but may also contribute to deterioration of long-term organ function.Indonesian Endowment Fund for Educatio