Extracellular miRNAs in saliva as a reflection of the body's response to particulate matter exposure in children

Abstract

Particulate matter (PM) is a mixture of small particles causing air pollution and affecting human health by inducing systemic inflammation and oxidative stress. Recently, PM was shown to affect miRNA expression in microvesicles. In this project, expression of 3 PM-sensitive miRNAs was evaluated in association with PM. Saliva was collected from 90 children at two different time points. Exosomal fraction from saliva was isolated using differential ultracentrifugation and total RNA enriched with small RNAs was isolated. cDNA of the miRNAs was synthesized using megaplex stem-loop primers for quantification of miR222, miR146a and miR155. The effect of different normalizers of the expression levels was studied. Multivariate-adjusted mixed models with correction for FDR were applied to estimate effects of PM exposure on miRNA expression. CD63-positive vesicular structures were found in the ultracentrifuged saliva samples, though vesicles were found larger than <100 nm. The use of a spike-in miRNA was shown to be the best normalizer. After multivariate-adjustment, an IQR increase in UFP was associated with a 21% (± 7%) increase in miR222 expression for indoor exposure during the study visit (P=0.025) and 34% (± 9%) for outdoor UFP concentrations (P=0.0054). In a univariate model between miR222 and blood pressure (BP), an association between could be found (P=0.0326 for systolic BP and P=0.0297 for diastolic BP). After adjustment for covariates only a trend was observed (P<0.1). We confirm the use of saliva for finding early al