An investigation into the relationships between novel Y chromosome-linked long non-coding RNAs and coronary artery disease

Abstract

Coronary artery disease (CAD) is the most common type of cardiovascular disease and is one of the leading causes of morbidity and mortality globally. However, the pathogenesis of atherosclerosis which leads to CAD and results in heart attacks, heart failure and death is not well understood. In this context, studies have demonstrated a positive correlation between increased hepatic free fatty acids (FFAs) in atherosclerosis and CAD. Although CAD has welldefined environmental risk factors, genome-wide association studies (GWAS) have demonstrated a genetic influence on CAD. Previous studies have shown that genetic variation within the human Y chromosome is associated with an increased risk of developing CAD independent of traditional cardiovascular risk factors; possibly through a modulating effect of an adaptive immunity and inflammatory response by macrophages in men. However, no Y chromosome-linked gene has been investigated in this disease so far. Long non-coding RNAs (lncRNAs) have recently gained focused attention as a new class of regulatory RNA molecules involved in cardiovascular function and associated disease, particularly long intergenic noncoding RNAs (lincRNAs), the largest class within the lncRNA group so far. To date, Y chromosome-linked lincRNAs are poorly characterised and the potential link between these non-coding RNA molecules and CAD in men has not been investigated. In this context, I hypothesised that Y chromosome-linked lncRNAs may regulate pathways involved in lipid metabolism and trigger an over accumulation of FFAs in coronary arteries contributing to atherosclerosis, the underlying cause of CAD. The main objective of this thesis was to therefore further investigate the relationship between the Y chromosome, lncRNAs and CAD in light of the deficiencies within the literature to better understand the causative molecular mechanisms of CAD pathophysiology in men. In my first study (Chapter 2), I identified for the first time through gene expression analysis (real-time PCR) the expression of the following (unannotated in PubMed) Y chromosomelinked lincRNA transcripts: lnc-KDM5D-4:1, lnc-ZFY-1:1, lnc-ZFY-1:3, lnc-ZFY-2:1, lnc- RBMY1B-1:1, lnc-RBMY1B-1:4, lnc-RBMY1J-1:1, lnc-RBMY1J-1:2, and lnc-RBMY1J- 1:3, across 21 different normal, human tissues such as adipose, bladder, brain, cervix, colon, esophagus, heart, kidney, liver, lung, ovary, placenta, prostate, skeletal muscle, small intestine, spleen, testes, thymus, thyroid, trachea, and white blood cells (WBCs) (leukocytes). I found that Y-linked lincRNAs were expressed at low levels (with the lowest CT number equal at 24.5) with a high tissue-specificity for some. Also, the Y-linked RNA gene lnc-KDM5D-4 was widely expressed across male tissues while the Y-linked RNA gene lnc-RBMY1J-1 was specific to the testes. Furthermore, this study presents the first evidence through gene expression analysis that the Y chromosome-linked lincRNA transcripts, lnc-KDM5D-4:1, lnc- ZFY-1:1, lnc-ZFY-1:3, lnc-ZFY-2:1, lnc-RBMY1B-1:1, lnc-RBMY1B-1:4, and lnc- RBMY1J-1:3 are expressed in male leukocytes. Hence, these lincRNAs could be potential non-protein coding gene candidates for CAD research. Knowing that the Y chromosome contributes to lipid levels in humans, to further explore the potential function of these Y-linked lincRNAs in CAD in men, I then studied their expression in a fatty liver context (steatosis-associated atherosclerosis) (Chapter 3). This was performed using the human hepatocellular liver carcinoma cell line, HepG2; the human model of liver cells in CAD research. This study showed for the first time that the Y-linked lincRNA transcripts lnc-KDM5D-4:1, lnc-ZFY-1:1, lnc-ZFY-2:1, lnc-RBMY1B-1:1, and lncxix RBMY1B-1:4 were expressed in HepG2 cells, hence in hepatocellular carcinoma (HCC). Furthermore, this study demonstrated that lnc-KDM5D-4 is a nuclear-retained lincRNA using RNA fluorescence in situ hybridisation (RNA FISH), and is upregulated in palmitate-induced steatosis within hepatocytes (Fold Change = 2.16; p-value = 0.00216). The human Atherosclerosis RT2 Profiler™ PCR Array determined that the silencing of lnc-KDM5D-4 in HepG2 cells was triggering the upregulation of the inhibitor of apoptosis (IAP) gene baculoviral IAP repeat containing 3 (BIRC3) (Fold Change = 12.45, p-value = 0.000025), a well-described protein-coding gene expressed by vascular smooth muscle cells and macrophage foam cells of the inflamed vascular wall of atherosclerotic arteries. Furthermore, perilipin 2 (PLIN2), a gene known to be implicated in lipid metabolism, was also found upregulated. Therefore, this study provides the first evidence for the involvement of a Ychromosome- linked lincRNA, lnc-KDM5D-4, in steatosis-associated atherosclerosis and its retained-nuclear cellular localisation in human hepatocytes, suggesting a function which takes place in the cell nucleus and may play a role in regulating metabolic processes in the liver that are implicated in atherosclerosis. Having shown that a Y chromosome-linked lincRNA could be involved in the determination of lipid level and hence atherosclerosis in men, and to further explore the role of lnc-KDM5D- 4, the expression of this Y-linked lincRNA was studied in human coronary artery smooth muscle cells, especially in atherosclerotic coronary artery cells (Chapter 4). The expression of other non-coding RNAs were also studied such as the protein kinase, Y-linked, pseudogene (PRKY) - previously considered as a new functional candidate for the development of CAD. By analysing the transcriptome of human atherosclerotic and non-atherosclerotic coronary artery smooth muscle cells, I established evidence for the implication of the human Y chromosome in atherosclerosis and CAD. This study exposed the general underexpression of the transcripts from the Y chromosome in atherosclerotic cells implicating a loss or a repression of this chromosome in relation to CAD. Furthermore, this research determined by RNA sequencing a significant downregulation of seven transcripts from Y chromosome genes, including RPS4Y1, USP9Y, DDX3Y, TXLNGY, NLGN4Y and PRKY. RNA FISH determined the subcellular localisation of PRKY in smooth muscle cells by showing a nuclear and a cytoplasmic expression. Furthermore, qPCR gene expression analysis demonstrated that lnc- KDM5D-4 is significantly downregulated in atherosclerotic cells in comparison to the nonatherosclerotic cells. Together, these results showed that lnc-KDM5D-4 is a potential regulator of PLIN2 and BIRC3 genes. Therefore, the downregulation of lnc-KDM5D-4 in atherosclerotic coronary artery smooth muscle cells suggests that this downregulation could be linked to the inflammation of the vascular smooth muscle cells in pathophysiology of CAD via the inhibition of apoptosis of the vascular smooth muscle cells triggered by the upregulation of BIRC3 in these cells. Overall, this study is the first to emphasise a potential involvement of a Y-specific lincRNA, called lnc-KDM5D-4, as a potential contributor to physiology in males. Lnc-KDM5D-4 knockdown resulted in an upregulation of anti-apoptosis and lipid metabolism-related genes. Collectively, our data suggest that the male–specific lnc-KDM5D-4 may regulate key processes in cellular inflammation that trigger atherosclerosis and CAD in men. Accordingly, this data suggests that lnc-KDM5D-4 may provide a novel molecular biomarker for atherosclerotic arteries, and could potentially lead to revolutionary treatment modalities on Y-linked lincRNA as therapeutic agents to manipulate CAD-causing genes in men.Doctor of Philosoph