Exosomal microRNAs are novel circulating biomarkers in cigarette, waterpipe smokers, E-cigarette users and dual smokers

Abstract

Background Electronic cigarettes (e-cigs) vaping, cigarette smoke, and waterpipe tobacco smoking are associated with various cardiopulmonary diseases. microRNAs are present in higher concentration in exosomes that play an important role in various physiological and pathological functions. We hypothesized that the non-coding RNAs transcript may serve as susceptibility to disease biomarkers by smoking and vaping. Methods Plasma exosomes/EVs from cigarette smokers, waterpipe smokers and dual smokers (cigarette and waterpipe) were characterized for their size, morphology and TEM, Nanosight and immunoblot analysis. Exosomal RNA was used for small RNA library preparation and the library was quantified using the High Sensitivity DNA Analysis on the Agilent 2100 Bioanalyzer system and sequenced using the Illumina NextSeq 500 and were converted to fastq format for mapping genes. Results Enrichment of various non-coding RNAs that include microRNAs, tRNAs, piRNAs, snoRNAs, snRNAs, Mt-tRNAs, and other biotypes are shown in exosomes. A comprehensive differential expression analysis of miRNAs, tRNAs and piRNAs showed significant changes across different pairwise comparisons. The seven microRNAs that were common and differentially expressed of when all the smoking and vaping groups were compared with non-smokers (NS) are hsa-let-7a-5p, hsa-miR-21-5p, hsa-miR-29b-3p, hsa-let-7f-5p, hsa-miR-143-3p, hsa-miR-30a-5p and hsa-let-7i-5p. The e-cig vs. NS group has differentially expressed 5 microRNAs (hsa-miR-224-5p, hsa-miR-193b-3p, hsa-miR-30e-5p, hsa-miR-423-3p, hsa-miR-365a-3p, and hsa-miR-365b-3p), which are not expressed in other three groups. Gene set enrichment analysis of microRNAs showed significant changes in the top six enriched functions that consisted of biological pathway, biological process, molecular function, cellular component, site of expression and transcription factor in all the groups. Further, the pairwise comparison of tRNAs and piRNA in all these groups revealed significant changes in their expressions. Conclusions Plasma exosomes of cigarette smokers, waterpipe smokers, e-cig users and dual smokers have common differential expression of microRNAs which may serve to distinguish smoking and vaping subjects from NS. Among them has-let-7a-5p has high sensitivity and specificity to distinguish NS with the rest of the users, using ROC curve analysis. These findings will pave the way for the utilizing the potential of exosomes/miRNAs as a novel theranostic agents in lung injury and disease caused by tobacco smoking and vaping.

Differential plasma exosomal long non-coding RNAs expression profiles and their emerging role in E-cigarette users, cigarette, waterpipe, and dual smokers

“…Based on our recent studies on profiling of miRNA in smokers, E-cig users, Waterpipe smokers and dual users [23], we compared the exosomal lncRNA profiles in the blood plasma from NS, CS, WP, CSWP and E-cig users. Genome-wide association studies have identified…”

“…Each subject filled a questionnaire containing information about demographics, clinical symptoms, E-cig use, and vaping history [ 25 , 26 ]. We collected 20–25 ml blood by venipuncture from smokers (CS), vapers (E-cig), waterpipe (WP) users, and subjects indulged in both cigarette smoking and waterpipe use (CSWP) as previously shown [ 23 , 26 ]. Subjects included for the study were between 18–65 yrs.…”

“…We employed commercially available kit from Norgen Biotek (Cat# 57400; Ontario, Canada) to isolate exosomes from human plasma. Plasma exosomes were isolated as per manufacturer’s protocols as described earlier [ 23 , 27 ]. In brief, 1 ml plasma was centrifuged at 400 g (~2000 rpm) for 2 min.…”

“…Transmission electron microscopy (TEM) was used to visualize the isolated exosomes and nanoparticle tracking analysis was performed to analyze the particle size and concentration as described [ 23 ].…”

“…At the same time, the prevalence of ‘hookah’ or waterpipe is also increasing [ 21 , 22 ]. Considering this and based on our recent observations of differential regulation of miRNA [ 23 ], we compared the expression profiles of plasma-derived exosomal lncRNAs from non-smokers (NS), cigarette smokers (CS), E-cig users (E-cig), waterpipe (WP) users and dual (cigarette and waterpipe) smokers (CSWP). The objective of this study was to characterize the lncRNA content of the plasma-derived exosomes from a representative population of these tobacco product users to help identify novel biomarkers, which may be relevant to the associated pulmonary pathologies, such as COPD, asthma, and IPF.…”