Veneruso, Iolanda (2025) RNA-seq Analysis of Human Atherosclerotic Plaques and their Adjacent Regions: a Focus on lncRNAs. [Tesi di dottorato]

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Abstract

Aims. Even if the role of long non-coding RNAs (lncRNAs) in several diseases are emerging, their impact on the development atherosclerosis is still under investigation. The aim of this study was to evaluate how lncRNAs play a role as promoter and biomarker of atherogenic process in humans. Materials and Methods. Carotid atherosclerotic plaques, their adjacent regions and plasma samples were collected from 15 patients underwent endarterectomy and processed for total RNA-Seq analysis by Next Generation Sequencing. Bioinformatic analysis was conducted on R. Plasma samples from other 35 atherosclerotic patients and from 19 healthy individuals (total of 50 cases and 19 controls) were collected. After circulating RNA extraction, quantitative real-time PCR (qRT-PCR) experiments were conducted with SYBR-Green PCR MasterMix using GAPDH as housekeeping gene. Statistical analysis was performed using SPSS software. Results. Among the 915 differentially expressed genes in plaques versus adjacent regions, lncRNA H19 resulted overexpressed in plaques (logFC=4.3 - p=0.00004) and present in plasma after sequencing. qRT-PCR results on tissues confirmed that lncRNA H19 is overexpressed in plaques samples [16.5 (6.5-40.5)] compared to adjacent regions [0.1 (0.07-1.8) - p=0.001]. Circulating lncRNA H19 levels were higher in plasma from atherosclerotic patients [1.15 (0.7-2.43)] than in plasma from healthy controls [0.7 (0.3-1) - p=0.004]. Higher levels of lncRNA H19 were found in patients with Gray-Weale grade 2 – lipid-reach plaques (p=0.022), in smokers (p=0.028) and in dyslipidemic patients (p=0.020). Gray-Weale resulted independently negatively associated with H19 levels (beta-coeff. = -0.498 – p=0.004). Conclusion. We reported the overexpression of lncRNA H19 in human atherosclerotic carotid plaques rather than in the adjacent lower-grade regions, confirming its involvement in the development of atherosclerotic plaques in humans. Higher levels of lncRNA H19 were found in plasma from atherosclerotic patients rather than in controls, associated with unstable plaques (Gray-Weale = 2), thus allowing to consider lncRNA H19 a good circulating biomarker (AUC=0.73) reflecting the presence of unstable atherosclerotic carotid plaque.

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