MicroRNA and mRNA Processing Variations in Normal and Hypertrophic Hearts

Abstract

Cardiovascular disease (CVD) affects one in six Australians, with healthcare expenditure that exceeds that for any other disease Recently, there has been an increased interest in studying the regulation of and by RNAs to find novel targets for the treatment of CVD. microRNAs (miRNAs) in particular, have been implicated in the pathogenesis of CVD, and thus have emerged as a promising candidate for therapeutic interventions. In addition to differential miRNA expression, miRNA-mediated control is also affected by variable miRNA processing and alternative 3’-end cleavage and polyadenylation (APA) of their mRNA targets. To what extent these phenomena play a role in the heart remains unclear. Thus, the major aim of this thesis is to uncover the expression and processing variations of miRNAs and mRNA 3’-ends, and evaluate their changes during cardiac hypertrophy.

Thoracic aortic constriction (TAC) was performed to induce hypertrophy in C57BL/6J mice. RNA extracted from cardiomyocytes of sham-treated, pre-hypertrophic (2 days post-TAC), and hypertrophic (7 days post-TAC) mice was subjected to small RNA- and poly(A)-test sequencing (PAT-Seq). The sequencing datasets were mined for expressions of non-coding and protein-coding transcripts and their variants.

The analyses of differential expression of miRNA and mRNA during cardiac hypertrophy matched expectations, although we identified ~400 mRNAs and hundreds of noncoding RNA loci as altered with hypertrophy for the first time. There is a widespread occurrence of miRNA processing variations in the cardiomyocyte, however their relative proportions remained largely unchanged during hypertrophy. Our mRNA 3’end-sequencing data identified novel 3’UTRs for 7,348 genes. Notably, there was a significant net shift towards shorter 3’UTR variants as hypertrophy develops. We independently validated several examples of 3’UTR proportion change, and showed that alternative 3’UTRs associate with differences in mRNA translation. We combined the small RNA- and PAT-sequencing datasets for a cardiac-specific miRNA target predictions analysis. Differential analyses of these interactions during hypertrophy suggested that the connectivity between miRNA and their 3’UTR targets are modulated, with an overall decrease in miRNA repression strength during cardiac hypertrophy. The findings presented in this thesis suggest that APA contributes to altered gene expression with the development of cardiomyocyte hypertrophy. This thesis provides a rich resource for a systems-level understanding of miRNA-mediated regulation in physiological and pathological states of the heart. Show more