Cystic Fibrosis (CF), is due to CF-transmembrane-conductance-regulator (CFTR) loss-of-function. Significant heterogeneity exists between patients, suggesting potential epigenetic regulation, and comorbidities develop with time. MiRNAs are non-coding RNAs that act as epigenetic regulators. Although many studies have focused on the role for miRNAs in regulating CFTR gene expression, little attention has been given to how CFTR influences their expression and how this affects growth, glucose metabolism and fertility.
We aimed to assess changes in miRNA expression levels dependent on CFTR loss-of-function, and to subsequently identify the affected molecular pathways.
CFBE41o-(homozygous for F508del mutation), and IB3(heterozygous for F508del/W1282X mutations) human immortalized cell lines were used since they reflect the most common genetic mutations in humans for CF, and 16HBE14o- (derived from normal bronchus) cells as controls. RNA was isolated from each cell line (miRVana kit). Global miRNA profiles were assessed using Taq-Man Array Human MicroRNA Card Set v3.0 which enable accurate quantitation of 754 human miRNAs. Relative quantification was performed using U6snRNA and RNU48 as endogenous controls and 16HBE14o- cells as calibrator for relative abundance of each miRNA which was calculated as fold change (log22-DDCt). MiRNAs showing fold-changes of ≥+2 or ≤-2 (P-value=0.05) were considered. MiRNA target genes and KEGG pathways were identified using miRWalk software and DIANA-mirPathv.3 web-server, respectively.
In CF cell lines, 41 miRNAs showed significant changes,17 were up-regulated and 24 down-regulated. Ten miRNAs were regulated in both CFBE41o- and IB3 cells, and a subset of 3 showed significant differences between the two CF cell lines (miR-200b, miR-616, miR-942). Further, 14 miRNAs were regulated in CFBE41o- only and 17 in IB3 only, suggesting genotype-specific effects.
The 41 miRNAs targeted genes within pathways involved with longitudinal growth (TGF-beta, mTOR, MAPK, Prolactin, and PI3K-Akt signaling pathways), glucose metabolism (Hippo signaling, Fatty acid biosynthesis, FoxO signaling) and fertility (Oocyte meiosis, Estrogen signaling, Progesterone-mediated oocyte maturation). Interestingly, within the growth regulating pathways, genes involved with genetic short stature in humans were highlighted (BRAF, FLNB, GNAS, NF1, PDE3A, RAF1, STAT5B, IGF1R) as well as genes involved with glucose metabolism dysregulation (FOXO1, FOXO3, G6PC, GSK3B, IRS1, IRS2, IRS4), and infertility (ESR1, PGR, Cyclins, BCL2, Caspases).
In conclusion, CFTR loss-of-function determines changes in the miRNA network and thus on gene regulation. Changes are related with the specific genetic mutations and have effects on genes involved with longitudinal growth, glucose metabolism and fertility which can show alterations in CF patients.
19 - 21 Sep 2019
European Society for Paediatric Endocrinology