C/D Box Small Nucleolar RNAs of the 14q32 Noncoding RNA Gene Cluster Play an Important Role in Human Cardiovascular Disease

We have previously shown that 14q32 microRNAs play a crucial role in cardiovascular disease. Besides 54 microRNAs, the human 14q32 locus also encodes 41 small nucleolar RNAs (snoRNAs). The 41 snoRNAs in the 14q32 locus are all orphan C/D box snoRNAs and their biological function as well as their role in human physiology and pathology is unknown. In patients with coronary artery disease, the vena saphena magna is often used for coronary bypass surgery, but unfortunately, such bypasses often fail due to restenosis. We compared 14q32 snoRNA expression in 8 healthy human venae saphenae magnae with that in 20 failed coronary bypasses. We measured expression of seven 14q32 snoRNAs, SNORD112, SNORD113.2, SNORD113.6, SNORD113.7, SNORD113.8, SNORD113.9, SNORD114.1, by rt/qPCR and found that all were upregulated in failed coronary bypasses. Striking were SNORD113.2 (17.3-fold upregulation) and SNORD113.9 (19.8-fold). For the 41 human 14q32 snoRNAs, there are only 7 known murine equivalents. However, sequence homology is high. We found that all murine 14q32 snoRNAs are also upregulated in failed murine bypasses. To further investigate the role of the 14q32 snoRNAs in human cardiovascular disease, we analyzed the data of a Genome Wide Association Scan (GWAS) in two study populations, the Genetic Determinants of Restenosis (GENDER) and the Prospective Study of Pravastatin in the Elderly at Risk (PROSPER), including 6110 individuals. From the snoRNA-encoding region, 42 single nucleotide polymorphisms (SNPs) were included in the GWAS, of which 17 were associated with cardiovascular disease. We found that there is no Linkage Disequilibrium between the snoRNA and microRNA regions, or between the snoRNA and MEG3 regions. This means that 14q32 orphan snoRNAs exert their effects on cardiovascular disease in humans independently of the 14q32 microRNAs. However, the mechanism-of-action through which they exert these effects remains to be determined. Using 3 rd generation antisense (3GA), we achieved up to 85% knockdown of snoRNA expression in vitro and ex vivo . Using 3GA technology in mouse models for cardiovascular disease will provide us the necessary tools to elucidate the mechanism-of-action through which 14q32 snoRNAs exert their effects in cardiovascular disease.