Circulating microRNA and isomiR Profiles in Patients with Varying CVD Severity: Using isomiRs as Potential Biomarkers and Therapeutic Targets
Authors: James Amanda Marie , Thomas Sheena , Rooney Kimberly , Searles Charles D 
Published: November 2023

MicroRNAs(miRNAs) are small, non‐coding RNAs that are post‐transcriptional regulators of gene expression. They are recognized for their roles both as modulators of disease progression and as biomarkers of disease activity in diseases such as neurological diseases, cancers, and cardiovascular diseases (CVD). CVD is a chronic inflammatory process that is associated with changes in intra‐ and extracellular miRNA abundance, and these circulating miRNA profiles are promising novel biomarkers for CVD severity. Many extracellular miRNAs are enriched in microvesicles (MV) and could potentially be therapeutic targets. We hypothesized that CVD‐associated variations in the circulating miRNA profile are related to: 1) changes in the abundance of miRNA isoforms (isomiRs),2) shifts in the isomiRs’ extracellular transport modality, and 3) alterations inMV counts based on CVD severity. IsomiR sequences are typically 1–3 nucleotides different from the consensus miRNA sequences but little is known about their biological function. Using RNA‐deep sequencing, the abundance/frequency of consensus miRNAs and isomiRs were assessed in 18 human blood samples: nine samples were from patients with only clinical risk factors (RFs) for CVD and nine samples were from patients diagnosed with significant CVD. From these analyses, 46 different miRNAs were identified as having both high frequency and abundance in these human blood samples; for 22 of these miRNAs, the expression of isomiRs were higher than that of the consensus miRNA. Among the 22 miRNAs that were isomiR‐dominant, 90% had a deletion orinsertion in the 3′ region with no changes in the seed sequence region. Using qRT‐PCRprimers that were custom‐designed for quantitation of each of the 22 dominant isomiRs, three isomiRs (isomiR‐10b, ‐93, and ‐181a) were determined to demonstrate statistical differences among patients (20 RFs; 24 CVD) with varying CVD severity and contained the consensus miRNA seed sequences. The abundance of isomiR‐10b was 10‐fold higher in the plasma of patients with significant CVD compared to those with RFs for CVD, whereas the abundance of isomiR‐181a was 10‐fold higher only in the MV fraction of plasma from patients with CVD compared to patients with only RFs. Lastly, isomiR‐93 was 1,000‐fold higher in the plasma of patients with CVD compared to RFs and a 100,000‐fold higher in the MVs of patients with CVD. Additionally, plasma MV counts were quantitated by flow cytometry. There was a significant increase in the number of MVs from patients with RFs for CVD compared to those with severe CVD. IsomiR abundance and frequency within certain extracellular transport modalities coupled with circulating MV counts could be used as biomarkers to predict CVD severity. Furthermore, these data give insight into why understanding the function of these isomiRs is essential; once the function is completely understood, these 3 isomiRs could be used as therapeutic targets for patients with CVD.