Connecting rules from paired miRNA and mRNA expression data sets of HCV patients to detect both inverse and positive regulatory relationships

© 2015 Song et al.; licensee BioMed Central Ltd. Background: Intensive research based on the inverse expression relationship has been undertaken to discover the miRNA-mRNA regulatory modules involved in the infection of Hepatitis C virus (HCV), the leading cause of chronic liver diseases. However, biological studies in other fields have found that inverse expression relationship is not the only regulatory relationship between miRNAs and their targets, and some miRNAs can positively regulate a mRNA by binding at the 5' UTR of the mRNA.Results: This work focuses on the detection of both inverse and positive regulatory relationships from a paired miRNA and mRNA expression data set of HCV patients through a 'change-to-change' method which can derive connected discriminatory rules. Our study uncovered many novel miRNA-mRNA regulatory modules. In particular, it was revealed that GFRA2 is positively regulated by miR-557, miR-765 and miR-17-3p that probably bind at different locations of the 5' UTR of this mRNA. The expression relationship between GFRA2 and any of these three miRNAs has not been studied before, although separate research for this gene and these miRNAs have all drawn conclusions linked to hepatocellular carcinoma. This suggests that the binding of mRNA GFRA2 with miR-557, miR-765, or miR-17-3p, or their combinations, is worthy of further investigation by experimentation. We also report another mRNA QKI which has a strong inverse expression relationship with miR-129 and miR-493-3p which may bind at the 3' UTR of QKI with a perfect sequence match. Furthermore, the interaction between hsa-miR-129-5p (previous ID: hsa-miR-129) and QKI is supported with CLIP-Seq data from starBase. Our method can be easily extended for the expression data analysis of other diseases.Conclusion: Our rule discovery method is useful for integrating binding information and expression profile for identifying HCV miRNA-mRNA regulatory modules and can be applied to the study of the expression profiles of other complex human diseases

Rule mining on microRNA expression profiles for human disease understanding

University of Technology Sydney. Faculty of Engineering and Information Technology.This research employs rule mining methods to study the important roles of miRNAs in human diseases. From past experience and from reviewing the literature, rule mining is a widely used data mining technique for the discovery of interesting relationships in large data sets. MicroRNAs (miRNAs) are endogenous and highly conserved non-coding RNA molecules. They can inhibit and/or promote the post-transcriptional expression of target messenger RNAs (mRNAs). miRNAs thus play a pivotal role in a cell’s differentiation, proliferation, growth, mobility, and apoptosis, as well as in viral replication and proliferation. This has inspired many research works aimed at detecting miRNAs’ functions in human disease. However, with the current deluge of miRNA data, previous works have suffered from limitations in terms of handling the relationship between various molecules. Firstly, they usually identify single miRNAs as biomarkers, and always produce low sensitivity and specificity. Secondly, intensive research largely depends on the inverse expression relationships between miRNAs and mRNAs to discover miRNA-mRNA regulatory modules. Finally, the miRNA-miRNA co-regulations and miRNA self-regulations have not been well investigated. As a result, rule mining is a powerful new technology with great potential to help researchers focus on the most important miRNAs for understanding human diseases. This thesis reports our past and current research outcomes in this area. The contributions of the thesis are as follows: • A novel rule mining method is proposed to detect the significant miRNA biomarkers. • A “change to change” method is proposed to mine both positive and negative regulatory relationships from paired miRNA and mRNA expression data sets. • A progressive data refining approach is proposed to identify the lung cancer miRNA-miRNA co-regulation network. • A novel framework is proposed to detect the self-regulation miRNAs. The research was conducted through four case studies. (1) The first case study was on lung squamous cell carcinoma for accurate diagnosis of this disease through the reliable miRNA biomarkers identified by a novel rule discovery method. (2) The second case study was on paired miRNA and mRNA expression data of HCV patients to detect both positive and negative regulatory modules. (3) The third case study was on lung cancer data sets for the computational methods to identify miRNA-miRNA co-regulation networks and miRNA-miRNA co-regulatory relationships. (4) The fourth case study was on multiple data types to infer self-regulation miRNAs in humans through an integrative rule mining framework and approach. All the results have been verified by the existing literature and databases

MicroRNAs miR-203-3p, miR-664-3p and miR-708-5p are associated with median strain lifespan in mice

This is the final version of the article. Available from Springer Nature via the DOI in this record.MicroRNAs (miRNAs) are small non-coding RNA species that have been shown to have roles in multiple processes that occur in higher eukaryotes. They act by binding to specific sequences in the 3' untranslated region of their target genes and causing the transcripts to be degraded by the RNA-induced silencing complex (RISC). MicroRNAs have previously been reported to demonstrate altered expression in several aging phenotypes such as cellular senescence and age itself. Here, we have measured the expression levels of 521 small regulatory microRNAs (miRNAs) in spleen tissue from young and old animals of 6 mouse strains with different median strain lifespans by quantitative real-time PCR. Expression levels of 3 microRNAs were robustly associated with strain lifespan, after correction for multiple statistical testing (miR-203-3p [β-coefficient = -0.6447, p = 4.8 × 10(-11)], miR-664-3p [β-coefficient = 0.5552, p = 5.1 × 10(-8)] and miR-708-5p [β-coefficient = 0.4986, p = 1.6 × 10(-6)]). Pathway analysis of binding sites for these three microRNAs revealed enrichment of target genes involved in key aging and longevity pathways including mTOR, FOXO and MAPK, most of which also demonstrated associations with longevity. Our results suggests that miR-203-3p, miR-664-3p and miR-708-5p may be implicated in pathways determining lifespan in mammals.This work was funded by the Wellcome Trust (grant number WT097835MF to D. Melzer and L.W. Harries), and the NIH-NIA (grant number AG038070 to The Jackson Laboratory)

Inference of gene regulation from expression datasets

The development of high throughput techniques and the accumulation of large scale gene expression data provide researchers great opportunities to more efficiently solve important but complex biological problems, such as reconstruction of gene regulatory networks and identification of miRNA-target interactions. In the past decade, many algorithms have been developed to address these problems. However, prediction and simulation of gene expression data have not yet received as much attention. In this study, we present a model based on stepwise multiple linear regression (SMLR) that can be applied for prediction and simulation of gene expression, as well as reconstruction of gene regulatory networks by analysis of time-series gene expression data, and we present its application in analysis of paired miRNA-mRNA expression data.Ph.D., Biomedical Engineering -- Drexel University, 201

Epitranscriptomic regulation in breast cancer and PCB-induced liver disease.

Post-transcriptional RNA modifications including N6-methyladenosine (m6A) regulate mRNA stability, splicing, and translation. My research examined m6A in two disease models: breast cancer (BCa) and non-alcoholic fatty liver disease (NAFLD). Acquired resistance to endocrine therapies (ET) develops in approximately 20% of BCa patients with estrogen receptor α positive (ER+) tumors following treatment. The mechanisms by which tumor cells evade ET are not completely understood. Using a cell line model, we investigated the role of an m6A reader protein, HNRNPA2B1 (A2B1) that is upregulated in ET-resistant ER+ BCa cells. Stable overexpression of A2B1 in ET-sensitive MCF-7 cells (MCF-7-A2B1), results in ET resistance, whereas knockdown of A2B1 in ET-resistant cells restored ET-sensitivity. microRNAs (miRNAs) downregulated by transient overexpression of A2B1 were identified to target two key enzymes (PSAT1 and PHGDH) in the serine biosynthetic pathway (SSP) which is upregulated in ET-resistant BCa cells and in tumors from patients with ET-resistant disease. Using luciferase assays, PSAT1 and PHGDH were validated as bona fide targets of miRNAs downregulated by A2B1 (miR-145-5p and miR-424-5p targeting PSAT1, miR-34b-5p and miR-876-5p targeting PHGDH). Exogenous overexpression of the validated miRNAs decreased endogenous PSAT1 and PHGDH in ET-resistant BCa cells, resulting in increased sensitivity to ET in vitro. In the second model, alterations in the m6A epitranscriptome were identified in the livers of male C57Bl/6Jmice after a single, oral exposure to polychlorinated biphenyls (PCB), a class of persistent organic pollutants, in combination with 12 weeks on a high fat diet (HFD). Our results demonstrated that exposure to PCBs in combination with a HFD resulted in major changes to the mRNA and miRNA transcriptomes, and m6A epitranscriptome. Pathway analysis of the genes in which m6A peaks were altered identified pathways involved in the progression from steatosis to steatohepatitis in NAFLD. PCB exposures also resulted in changes to alternative splicing (AS) mechanisms and events, suggesting that PCB-induced m6A changes contribute to altered isoforms expression in NAFLD. Taken together, the results in this dissertation demonstrate the significant role of altered m6A in two common human diseases

Changes in RNA regulatory processes during mammalian ageing

Ageing is defined as a system-wide, gradual loss in overall organ and tissue function across the lifespan of an organism, and in humans is the single largest risk factor for most chronic diseases. Thanks to ongoing improvements in healthcare, human life expectancy is steadily rising, but the proportion of life spent free of chronic disease (known as healthspan) is not extending concurrently in our increasingly aged population. Socio-economic costs are growing, both in terms of healthcare spending and quality of life. A central goal of ageing research therefore is to find methods of extending healthspan. However, ageing is a complex, heterogeneous process and the underlying mechanisms of ageing and determinants of lifespan/healthspan are still not well understood. RNA regulators of gene expression are important factors in the ageing process, and I hypothesise that they may have potential to affect healthspan, or act as biomarkers of ageing. In this thesis, I have examined some of these RNA regulatory factors and their associations with ageing and lifespan in mammals. In order to do this, I assessed the expression patterns of RNA regulatory factors in two mouse models and a human cohort. In one mouse model, I found that both mRNA splicing regulatory factors and microRNAs are associated with strain-specific longevity during normal ageing, and that it is possible that these regulators play a causal role in determining strain lifespan. In the second mouse model, I showed these splicing factors to be associated with dietary restriction (a known treatment for extension of lifespan) and provided evidence that they could be mechanistically involved in the lifespan response to dietary restriction. I also showed that expression levels of these splicing factors were associated with cognitive decline and reduction in physical ability in humans. These results indicate that correct RNA regulation is a key component of the ageing process and suggests that the factors that govern these processes may represent useful future targets for healthpan intervention in ageing people.Velux Foundatio

The Complex Network of miRNA and mRNA Target Interactions in Pancreatic Cancer

Pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) is one of the most lethal tumour types world-wide. The majority of patients present late with locally advanced or metastatic disease. Therefore, despite advances in operative techniques, perioperative management and oncological treatments, the overall 5-year survival remains <5%. Determining tumoural factors that contribute towards its aggressive nature may help in identifying novel molecular biomarkers and/or therapeutic targets. MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate target gene expression and are able to act as tumour suppressors or oncogenes. MiRNAs have been extensively profiled and implicated in the initiation and progression of PDAC. Furthermore, there is a possibility of translating miRNAs into clinically useful biomarkers. Here, I developed upon these initial observations and demonstrate that miRNAs can be used to differentiate low risk pancreatic benign cystic tumours (BCTs) from PDAC. We confirmed that these miRNAs regulate the expression of known PDAC oncogenes, and that miR-16, miR-126 and let-7d target BCL2, CRK and KRAS respectively. Next, in order to investigate the main contributors to tumourigenesis, an integrated molecular analysis (miRNA-mRNA) was performed in PDAC. By using a combination of network-based bioinformatics, miR-21, miR-23a and miR-27a were prioritised as important in PDAC progression. We demonstrated that the use of a combination of miRNA inhibitors (against miR-21, miR-23a and miR-27a) in a murine subcutaneous PDAC xenograft model was able to reduce tumour growth, better than oncomiR-21 inhibition alone. BTG2 and NEDD4L were found to be direct targets of the miRNA combination and were established as new candidate tumour suppressors in PDAC. The clinical relevance of this 3 miRNA signature was demonstrated, as high expressors of the combination have poor overall survival after surgical resection, independent of other clinicopathologic factors. Together, these studies identify specific miRNAs as important regulators of PDAC tumourigenesis and their possible use as biomarkers.Open Acces

Nucleic Acid Architectures for Therapeutics, Diagnostics, Devices and Materials

Nucleic acids (RNA and DNA) and their chemical analogs have been utilized as building materials due to their biocompatibility and programmability. RNA, which naturally possesses a wide range of different functions, is now being widely investigated for its role as a responsive biomaterial which dynamically reacts to changes in the surrounding environment. It is now evident that artificially designed self-assembling RNAs, that can form programmable nanoparticles and supra-assemblies, will play an increasingly important part in a diverse range of applications, such as macromolecular therapies, drug delivery systems, biosensing, tissue engineering, programmable scaffolds for material organization, logic gates, and soft actuators, to name but a few. The current exciting Special Issue comprises research highlights, short communications, research articles, and reviews that all bring together the leading scientists who are exploring a wide range of the fundamental properties of RNA and DNA nanoassemblies suitable for biomedical applications