The circulating IsomiR-ome of increased cardiovascular risk in patients with non-alcoholic fatty liver disease

Nataly Makarenkov ^1,2 Uri Yoel ^1,3 Yulia Haim ¹ Nikhil Bhandarkar S. ¹ Aryeh Shalev ⁴ Ilan Shelef ⁵ Idit Liberty F. ⁶ Gal Ben-Arie ⁵ David Yardeni ⁷ Assaf Rudich ¹ Ohad Etzion ⁷ Isana Veksler-Lublinsky ²

¹Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion university of the Negev, Israel
²Department of Software & Information Systems Engineering, Faculty of Engineering, Ben-Gurion university of the Negev, Israel
³The Endocrinology unit, Soroka University Medical Center, Israel
⁴Cardiology, Soroka University Medical Center, Israel
⁵Imaging, Soroka University Medical Center, Israel
⁶Diabetes clinic, Soroka University Medical Center, Israel
⁷Gastroenterology clinic, Soroka University Medical Center, Israel

Cardiovascular (CV) disease is the leading cause of mortality in patients with non-alcoholic-fatty-liver disease (NAFLD). Yet, as NAFLD is an independent risk factor for CV disease, traditional CV-risk-factors and clinically-based scoring systems poorly identify NAFLD patients with severely-elevated CV-risk. Here we hypothesize that in-depth analysis of circulating miRNAs can identify strong biomarkers for CV-risk in NAFLD.

Liver fat content was measured with magnetic-resonance-spectrometry (MRS); CV-risk was determined by traditional biomarkers, and by cardiac-CT measurement of coronary artery calcium (CAC) score. Plasma miRNAs were sequenced by next-generation-sequencing (NGS). N=13 patients, age>45y, with liver MRS-measured fat content≥5%(wt/wt) and free of overt CVD, were assessed for traditional CV-risk factors, and for CAC score-based CV-risk percentile (CAC-CV%). NGS identified 2,731 miRNAs and 404,011 different isomiRs, of which 280 and 1,418, respectively, passed an abundance threshold. Sixteen/two circulating miRNAs correlated positively/negatively, respectively, with CAC-CV%, 9 of which significantly discriminated high/low CV-risk by ROC-AUC-analysis. Three of these 18 miRNAs are unknown to associate with CVD, but their validated targets exhibit multiple enriched pathways related to CVD pathogenesis. isomiR-ome analyses uncovered 69 isomiRs highly correlated (R≥0.55) with CAC-CV%. Specific isomiRs of miR-101-3p, miR-144-3p, miR-421 and miR-484 exhibited stronger association with CAC-CV% compared to their respective reference-miRNA. Additionally, while miR-140-3p, miR-223-3p, miR-30e-5p and miR-342-3p did not correlate with CAC-CV%, specific seed-altered isomiRs exhibited strong correlation with coronary atherosclerosis burden, and their targets were uniquely enriched in CVD-related pathways. Exploring the circulating isomiR-ome holds promise for identifying novel biomarkers and potential mediators of increased CV-risk in patients with NAFLD.