Precision-cut lung slices (PCLS) have gained increasing interest as a model to study lung biology/disease and screening novel therapeutics. In particular, PCLS derived from human tissue can better recapitulate some aspects of lung biology/disease as compared with animal models. Several experimental readouts have been established for use with PCLS, but obtaining high-yield and -quality RNA for downstream analysis has remained challenging. This is particularly problematic for utilizing the power of next-generation sequencing techniques, such as RNA-sequencing (RNA-seq), for nonbiased and high-throughput analysis of PCLS human cohorts. In the current study, we present a novel approach for isolating high-quality RNA from a small amount of tissue, including diseased human tissue, such as idiopathic pulmonary fibrosis. We show that the RNA isolated using this method has sufficient quality for RT-qPCR and RNA-seq analysis. Furthermore, the RNA-seq data from human PCLS could be used in several established computational pipelines, including deconvolution of bulk RNA-seq data using publicly available single-cell RNA-seq data. Deconvolution using Bisque revealed a diversity of cell populations in human PCLS, including several immune cell populations, which correlated with cell populations known to be present and aberrant in human disease.

Non-coding RNAs (ncRNAs) are involved in the regulation of numerous biological processes and pathways and therefore have been extensively studied in human diseases. Previous reports have shown that non-coding RNAs play a crucial role in the pathogenesis and aberrant regulation of respiratory diseases. The altered expression of microRNAs (miRNAs) and long non-coding RNAs in blood and also locally in sputum or exhaled breath condensate influences lung function, immune response, and disease phenotype and may be used for the development of biomarkers specific for airway disease. In this review, we provide an overview of the recent works studying the non-coding RNAs in airway diseases, with a particular focus on chronic respiratory diseases of childhood. We have chosen the most common chronic respiratory condition—asthma—and the most severe, chronic disease of the airways—cystic fibrosis. Study of the altered expression of non-coding RNAs in these diseases may be key to better understanding their pathogenesis and improving diagnosis, while also holding promise for the development of therapeutic strategies using the regulatory potential of non-coding RNAs.

MicroRNAs are small non-coding RNAs that regulate immune response and inflammation. We assumed that miRNAs may be involved in the immune response during cystic fibrosis pulmonary exacerbations (CFPE) and that altered expression profile in the airways and blood may underlie clinical outcomes in CF pediatric patients. Methods: We included 30 pediatric patients diagnosed with cystic fibrosis. The biologic material (blood, sputum, exhaled breath condensate) was collected during pulmonary exacerbation and in stable condition. The miRNA expression profile from blood and sputum (n = 6) was done using the next-generation sequencing. For validation, selected four miRNAs were analyzed by qPCR in exosomes from sputum supernatant and exhaled breath condensate (n = 24). NGS analysis was done in Base Space, correlations of gene expression with clinical data were done in Statistica. Results: The miRNA profiling showed that four miRNAs (miR-223, miR-451a, miR-27b-3p, miR-486-5p) were significantly altered during pulmonary exacerbation in CF patients in sputum but did not differ significantly in blood. MiRNA differently expressed in exhaled breath condensate (EBC) and sputum showed correlation with clinical parameters in CFPE. Conclusion: MiRNA expression profile changes in the airways during pulmonary exacerbation in CF pediatric patients. We suggest that miRNA alterations during CFPE are restricted to the airways and strongly correlate with clinical outcome.

Lithium has been the most important mood stabilizer used for the treatment of bipolar disorder and prophylaxis of manic and depressive episodes. Despite long use in clinical practice, the exact molecular mechanisms of lithium are still not well identified. Previous experimental studies produced inconsistent results due to different duration of lithium treatment and using animals without manic-like or depressive-like symptoms. Therefore, we aimed to analyze the gene expression profile in three brain regions (amygdala, frontal cortex and hippocampus) in the rat model of mania and depression during chronic lithium administration (2 and 4 weeks). Behavioral changes were verified by the forced swim test, open field test and elevated maze test. After the experiment, nucleic acid was extracted from the frontal cortex, hippocampus and amygdala. Gene expression profile was done using SurePrint G3 Rat Gene Expression whole transcriptome microarrays. Data were analyzed using Gene Spring 14.9 software. We found that chronic lithium treatment significantly influenced gene expression profile in both mania and depression models. In manic rats, chronic lithium treatment significantly influenced the expression of the genes enriched in olfactory and taste transduction pathway and long non-coding RNAs in all three brain regions. We report here for the first time that genes regulating olfactory and taste receptor pathways and long non-coding RNAs may be targeted by chronic lithium treatment in the animal model of mania.

Children with cystic fibrosis (CF) suffer from chronic inflammation and recurrent pulmonary exacerbations (PEs). We aimed to test whether a specific miRNA could be associated with the occurrence of PE. We sequenced extracellular vesicle (EV)-derived miRNA in sputum (n = 20), exhaled breath condensate (EBC) (n = 11), and serum (n = 8) samples from pediatric patients during PE and the stable stage of CF. Four miRNAs: let-7c, miR-16, miR-25-3p and miR-146a, have been selected for validation in a larger group with reverse transcription quantitative real-time PCR (RT-qPCR) in sputum and serum, or droplet digital PCR (ddPCR) in EBC. Next-generation sequencing (NGS) differential expression analysis was done in Base Space, and the correlation between miRNAs expression and clinical data was calculated with Statistica. Functional annotation of selected miRNAs and their potential target genes was performed with miRDip and DAVID software. There were no differences in miRNA expression between stable and exacerbation in sputum and in serum. Validation of four selected miRNAs showed significant downregulation of miR-146a in serum. A panel of all four miRNAs (peripherally) was the best predictive model of exacerbation (p < 0.001, AUC = 0.96). Expression of airway miR-25-3p improved the diagnostic value of FEV1% pred and FVC% pred, while peripheral miR-146a improved the predictive model of C-reactive protein and neutrophilia. In silico analysis revealed a potential role for selected miRNAs in regulating processes associated with inflammation and tissue remodeling. We demonstrated that EVs contained in peripheral blood as well as local biomaterials can act as carriers for miRNAs with the diagnostic potential of predicting exacerbation in pediatric CF.