Identifying Candidate Circulating RNA Markers for Coronary Artery Disease by Deep RNA-Sequencing in Human Plasma

Abstract

Advances in RNA sequencing (RNA-Seq) have facilitated transcriptomic analysis of plasma for the discovery of new diagnostic and prognostic markers for disease. We aimed to develop a short-read RNA-Seq protocol to detect mRNAs, long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) in plasma for the discovery of novel markers for coronary artery disease (CAD) and heart failure (HF). Circulating cell-free RNA from 59 patients with stable CAD (half of whom developed HF within 3 years) and 30 controls was sequenced to a median depth of 108 paired reads per sample. We identified fragments from 3986 messenger RNAs (mRNAs), 164 long non-coding RNAs (lncRNAs), 405 putative novel lncRNAs and 227 circular RNAs in plasma. Circulating levels of 160 mRNAs, 10 lncRNAs and 2 putative novel lncRNAs were altered in patients compared with controls (absolute fold change >1.2, p < 0.01 adjusted for multiple comparisons). The most differentially abundant transcripts were enriched in mRNAs encoded by the mitochondrial genome. We did not detect any differences in the plasma RNA profile between patients who developed HF compared with those who did not. In summary, we show that mRNAs, lncRNAs and circular RNAs can be reliably detected in plasma by deep RNA-Seq. Multiple coding and non-coding transcripts were altered in association with CAD, including several mitochondrial mRNAs, which may indicate underlying myocardial ischaemia and oxidative stress. If validated, circulating levels of these transcripts could potentially be used to help identify asymptomatic individuals with established CAD prior to an acute coronary event.

A Comprehensive Review on Circulating cfRNA in Plasma: Implications for Disease Diagnosis and Beyond

“…In infectious diseases, the presence of pathogen-specific RNAs in plasma cfRNA can aid in the identification of the causative agent [116,117]. Furthermore, cfRNA analysis has potential applications in cardiovascular diseases [47,118,119] and neurodegenerative conditions [120]. The detection and analysis of cfRNA as a potential biomarker in disease diagnosis can provide valuable insights into the development and treatment responses of diseases.…”

Integrated analysis of circulating cell free nucleic acids for cancer genotyping and immune phenotyping of tumor microenvironment

“…The fragments from 160 of 3,986 mRNA, 10 of 164 lncRNAs and 2 of 405 novel lncRNAs were observed to be altered in patients with disease. The transcripts that were differentially elevated were enriched in mRNA that is coded by mitochondrial DNA that stipulates myocardial ischaemia and oxidative stress ( Ward et al, 2022 ).…”

cRNAsp12 Web Server for the Prediction of Circular RNA Secondary Structures and Stabilities

“…With the development of high-throughput sequencing technology and promising bioinformatics tools, emerging evidence indicates that circRNAs are stable, widely expressed, and play multifunctional roles in life processes, such as miRNA sponges, regulators of gene splicing and transcription, RNA-binding protein sponges and protein/peptide translators [ 1 , 2 , 3 , 4 , 5 , 6 ]. In consideration of their association with diseases [ 7 , 8 , 9 , 10 , 11 , 12 , 13 ], circRNAs have become a new hotspot in the clinical and research fields. Therefore, the accurate modeling of circRNAs annotation, structure determination and interaction analysis has a far-reaching impact on our understanding of their functions and our ability to develop RNA-based therapeutics [ 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 ].…”