Extracellular Vesicle-Enclosed Oxidative Stress- and Inflammation-Related microRNAs as Potential Biomarkers of Vitamin D Responsivity: A Pilot Study on Inflammatory Bowel Disease Patients with or without COVID-19

Abstract

The relationship between serum 25-hydroxyvitamin D (25(OH)D) levels, genomic response to vitamin D (Vit.D), and positivity to SARS-CoV-2 remains understudied. In this pilot study, during the follow-up of patients with Inflammatory Bowel Disease (IBD) and COVID-19, we investigated this issue by analyzing the molecular contents of serum extracellular vesicles (EVs) from six groups of IBD patients (n = 32), classified according to anti-SARS-CoV-2 status, 25(OH)D level, and Vit.D supplementation, by small RNA-seq. This analysis revealed differentially expressed miRNAs, PIWI-RNA, transfer RNA, small nucleolar RNAs, and protein-coding RNAs in the EVs obtained from these cohorts of IBD patients. Experimental validation evidenced a statistically significant increase in miR30d-5p, miR150-5p, Let-7f-5p, and Let-7a-5p in the anti-SARS-CoV-2-positive and low 25(OH)D and Vit.D supplemented groups with respect to the non-Vit.D supplemented group, indicating their responsiveness to Vit.D treatment. Bioinformatics analysis highlighted the regulation of these validated miRNAs by oxidative stress and inflammation, hallmarks of IBD and COVID-19. Our study reports an unprecedented panel of circulating EV-enclosed inflammation- and oxidative stress-related miRNAs, the potentiality of which, as biomarkers for Vit.D responsivity in IBD patients, needs to be explored in future studies on larger cohorts in order to allow clinicians to optimize current treatment strategies upon viral infection.

Keywords: COVID-19; Crohn’s disease; SARS-CoV-2; bioinformatics; biomarkers; extracellular vesicles; inflammation; inflammatory bowel diseases; oxidative stress; ulcerative colitis; vitamin D.

Figure 1

IBD patients 25(OH)D levels and serum EV characterization. (A). Plasma 25(OH)D levels are shown for the study cohort divided into groups (A, n = 4; B, n = 8; C, n = 7; D, n = 8; E, n = 4; F, n = 5). (B). Serum EV characterization by Western blotting; a representative blot is shown. (C). Nanosight tracking analysis of serum EVs; representative graphs are shown. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 2

Heatmap of miRNAs differentially expressed among EVs of the different groups of IBD patients under study. Table S6 shows mapping_Samples_ID.

Figure 3

QRT-PCR analysis of miRNAs differentially expressed in the EVs of IBD patients. * p < 0.05; ** p < 0.01. miR122-5p was selected as internal housekeeping miRNA. A-F are the groups of IBD patients.