MicroRNAs (miRs) play important roles in regulating cell function, with more than half of mammalian mRNAs under selective pressure to maintain pairing to miRs. However, with the exception of cancer, little is known regarding miR functions in the lung, and less known about miR expression and functions in airway epithelia. Cystic fibrosis (CF) is caused by mutations in the CFTR gene, a nucleotide regulated anion channel. CFTR is a low abundance mRNA, and loss of function has profound consequences. There is no knowledge of miR function in this disease. We surveyed miR expression in cultured well-differentiated human CF and non-CF airway epithelia and identified four miRs with increased expression in CF cells. Two miR targets of interest that we validated in pilot studies are SIN3A, a transcriptional co-repressor known to interact with regulatory elements in the CFTR promoter, and the CFTR gene product itself. We hypothesize that changes in miR expression resulting from CFTR mutations may contribute to the complex CF lung disease phenotype. There are three Aims:
Aim 1. To validate selected mRNA targets of differentially expressed miRs. The transcriptional regulatory gene SIN3A is a predicted miR-138 and -661 target, while CFTR is a target for miRs-494, -509-3p, and -661. We hypothesize that CFTR protein abundance is regulated by miRs at the transcriptional and translational levels. We will confirm the ability of differentially expressed miRs to bind the SIN3A and CFTR 3'UTRs in fusion gene (luciferase) assays. These and additional confirmed targets will be studied in gene specific studies.
Aim 2. To examine effects of miRs in the context of airway epithelia. Confirmed targets of miRs-138, -494, -509-3p, -661 will be assessed in gain and loss of function experiments in airway epithelia. For gain-of-function effects on SIN3A and CFTR, we will individually express miRs-138, - 494, -509-3p, -661 in CF and non-CF airway epithelia, and quantitatively assess endogenous SIN3A and CFTR mRNA and protein abundance. For loss of function effects, we will deliver anti-miRs to cells to inhibit interactions of endogenous miRs and their target 3'UTRs, and similarly assess mRNA and protein abundance of targets. For miRs with confirmed CFTR regulation we will further investigate the how miR expression alters cAMP-activated CFTR Cl- channel function in vitro. Similar strategies will be used for other priority targets.
Aim 3. To perform expanded expression profiling for a comprehensive discovery of differentially expressed miRs and their target mRNAs in CF and non-CF epithelia. We will use qRT PCR arrays to investigate miR expression patterns in a larger sample of CF (?F508/?F508) and non-CF primary human airway epithelia. MiRs with differential expression in CF will be confirmed by qRT PCR and Northern blot. In addition, CF and non-CF samples used in miR profiling will be profiled by Illumina Solexa mRNA sequencing (mRNA-seq) for their global gene expression patterns. Completion of this aim will provide a robust expression profile for 685 human miRs, expand our genomic efforts and miR target selection through anti-correlations with mRNA expression levels.
MicroRNAs are a class of small non-coding RNAs that play key roles in regulating gene expression. This project will investigate changes in microRNA expression in cystic fibrosis (CF) airway epithelia and how such changes contribute to the complex lung disease phenotype in CF. This will lead to a better understanding of cellular processes that may influence CF disease states, and could provide novel drug targets for therapeutic development.
| Ramachandran, Shyam; Osterhaus, Samantha R; Parekh, Kalpaj R et al. (2016) SYVN1, NEDD8, and FBXO2 Proteins Regulate ?F508 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Ubiquitin-mediated Proteasomal Degradation. J Biol Chem 291:25489-25504 |
