Small RNA-sequence analysis of plasma-derived extracellular vesicle miRNAs in smokers and patients with chronic obstructive pulmonary disease as circulating biomarkers

ABSTRACT

Extracellular vesicles (EVs) play a vital role in normal lung physiology to maintain homeostasis in the airways via intercellular communication. EVs include exosomes and microvesicles, and are characterized by their phospholipid bilayers. EVs have been recognized as novel circulating biomarkers of disease, which are released by different cell types. In this study, we used different EV isolation and purification methods to characterize the plasma-derived EV miRNAs from non-smokers, smokers and patients with COPD. A small RNA sequencing (RNA-seq) approach was adapted to identify novel circulating EV miRNAs. We found that plasma-derived EVs from non-smokers, smokers and patients with COPD vary in their size, concentration, distribution and phenotypic characteristics as confirmed by nanoparticle tracking analysis, transmission electron microscopy, and immunoblot analysis of EV surface markers. RNA-seq analysis confirmed the most abundant types of small RNAs, such as miRNAs, tRNAs, piRNAs snRNAs, snoRNAs and other biotypes in plasma-derived EVs. We mainly focused on miRNAs as novel biomarkers in smokers and patients with COPD for further analysis. Differential expression by DESeq2 identified distinct miRNA profiles (up-regulated: miR-22-3p, miR-99a-5p, miR-151a-5p, miR-320b, miR-320d; and down-regulated: miR-335-5p, miR-628-3p, miR-887-5p and miR-937-3p) in COPD versus smokers or non-smokers in a pairwise comparison. Gene set enrichment analysis (GSEA) of differentially expressed miRNAs revealed the top pathways, gene ontology and diseases associated with smokers and patients with COPD. We selectively validated miRNAs in EVs isolated from BEAS-2B cells treated with cigarette smoke extract by quantitative PCR analysis. For the first time, we report that plasma-derived EV miRNAs are novel circulating pulmonary disease biomarkers. Thus, molecular profiling of EV miRNAs has great translational potential for the development of biomarkers that may be used in the diagnosis, prognosis, and therapeutics of COPD.

KEYWORDS:

• Extracellular vesicles
• next-generation sequencing
• miRNAs
• biomarker
• COPD

Acknowledgments

This study was supported in part by the University of Rochester’s Lung Biology Strategic Plan - Pilot Project (IKS) and the National Institute of Health NIH 2R01HL085613, HL137738, HL135613, and ES028006 (IR). Dr Li’s effort is in part supported by the University of Rochester CTSA award number UL1 TR002001 from the National Center for Advancing Translational Sciences of the National Institutes of Health. Authors would like to thank Ms Daria Krenitsky and Dr Krishna Maremanda for their technical help and Ms Samantha R. McDonough, BS for her help in editing the manuscript.

Author contributions

IKS and IR: conceived and designed the experiments; IKS: conducted the experiments; IKS and DL: performed data analysis; IKS: wrote the manuscript; IKS, DL, and IR: edited the manuscript.

Conflicting interests

The authors have declared that no conflict of interest exists.

Supplementary material

The supplementary material for this article can be accessed here.

Additional information

Funding

This work was supported by the National Heart, Lung, and Blood Institute [HL135613], [2R01HL085613] and National Institute of Environmental Health Sciences [ES028006].