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[…] ression (both univariate and multivariate) was performed using R [] to identify miRNA (miR cycle threshold or CT value) associated with the pulmonary function phenotype of interest, log2 PC20. A least squares multivariate linear regression model including miR CT value, age, sex, and height was also calculated for each miRNA. A sensitivity analysis to assess outlier influence, and non-parametric models was also performed. The p-values were corrected using the Benjamini and Hochberg false discovery rate (FDR)., The miRNA dataset is available at the NCBI Gene Expression Omnibus (GEO, http://www.ncbi.nih.gov/geo/) GSE74770., A regulatory network between miRNA and genes was created with usage of Cytoscape (http://www.cytoscape.org/) [] and CyTargetLinker (http://projects.bigcat.unimaas.nl/cytargetlinker/) [] with Regulatory Interaction in Network Analysis (RegIN) miRTarBase release 6.1 (http://projects.bigcat.unimaas.nl/cytargetlinker/regins/regins-mirtarbase/) []. The Database for Annotation, Visualization and Integrated Discovery (DAVID, Version 6.8 (10/2016), https://david.ncifcrf.gov/home.jsp [] was used for KEGG [, ] pathway analysis and gene ontology., Functional validation of two significant miRNA was performed in human airway smooth muscle (HASM) cells as previously detailed []. The cells were transfected with 10nM of either scramble control (AllStars Negative Control siRNA, Qiagen) or miR mimic (Qiagen) using RNAiMax (Life Technology) according to manufacturer’s protocol. Seventy-two hours after transfection, cells were trypsinized for 8 minutes and then measured for both cell number and cell size by Moxi Z Cell Analyzer (Orflo). Cell growth was p […]

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