Elucidating the role of the long noncoding RNA, Gtl2, in rodent models of cardiac disease
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Recently, the discovery of noncoding RNAs (ncRNAs), such as long noncoding RNAs (lncRNAs) has altered the traditional view of gene regulation. Sequencing of genomes has brought to light the vast stretches of non-protein coding DNA regions that transcribe non-protein coding RNA. LncRNAs are multifunctional and extremely diverse. They can act as signals, decoys, scaffolds, guides, or enhancers. Several lncRNAs, such as Fendrr and Bvht, have been found to have important regulatory functions in cardiac disease and development. The Glt2-Dio3 locus, which is enriched in cardiac muscle, harbors two long intragenic RNAs, MEG3 and MEG8, and harbors one of the largest mammalian miRNA clusters. MEG3, which is termed Gtl2 in rat and mouse, contain 10 exons that are alternatively spliced and give rise to several variants. Gtl2 is conserved across human, rat, and mouse, which makes it an ideal candidate for research and a possible target for therapies. Based on the growing evidence for lncRNAs playing a role in cardiac muscle and our research on the Gtl2-Dio3 microRNAs (miRNAs), I focused on investigating the Gtl2 lncRNA in the heart. Antisense oligonucleotides (GapmeRs) were used to knockdown Gtl2 lncRNA expression levels in cultured, primary neonatal cardiomyocytes in basal and hypertrophic conditions. Although Gtl2 was effectively knocked down in basal conditions I was unable to achieve efficient knockdown in hypertrophic cardiomyocytes induced by phenylephrine treatment. Consequently, I did not observe any modulation of hypertrophy as determined by changes in the expression of Nppa and Nppb, established markers of cardiomyocyte hypertrophy.. Next, I utilized short hairpin RNA (shRNA) to knockdown Gtl2 lncRNA expression levels and obtained robust knockdown. Lastly, I designed a cardiac tropic adeno-associated virus 9 (AAV9) encoding MEG3 DNA for in vivo overexpression experiments as well as an adenovirus encoding MEG3 for in vitro overexpression experiments. These reagents will provide valuable resources for dissecting the functions of the Gtl2 lncRNA. Studies investigating the roles of Gtl2 in the diseased heart my lead to the development of other potential therapies to treat cardiac disease.