Synergistic post-transcriptional regulation of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) by miR-101 and miR-494 specific binding

Abstract

microRNAs (miRNAs) are a class of regulatory small non-coding molecules that control gene expression at post-transcriptional level. Deregulation of miRNA functions affects a variety of biological processes also involved in the etiology of several human mendelian and complex diseases. Recently, aberrant miRNA expression has been observed in Cystic Fibrosis (CF), an autosomal-recessive genetic disorder caused by mutations in the CFTR gene, in which a genotype-phenotype correlation is not always found. In order to determine miRNA role in CFTR post-transcriptional regulation, we searched for miR-responsive elements in the CFTR 3'-UTR. In silico analysis, performed using different computational on-line programs, identified some putative miRNAs. Both miR-101 and miR-494 synthetic mimics significantly inhibited the expression of a reporter construct containing the 3'-UTR of CFTR in luciferase assays. Interestingly, miR-101/miR-494 combination was able to markedly suppress CFTR activity by approximately 80% (p<0.001). This is one of the first in vitro studies implicating microRNAs as negative regulators of the CFTR gene expression. miRNA aberrant expression and function might explain the wide phenotypic variability observed among CF patients.

Figure 1. Bioinformatic prediction of microRNAs potentially targeting the CFTR gene.

(A) Schematic representation of the CFTR 3’UTR and miRNAs (triangles). Ribonucleotide sequences of the putative miR-101 and miR-494 responsive elements in aligned human, rhesus and mouse CFTR 3’UTRs paired with the mature human miR-sequence (from TargetScan 5.1 database). Numbers indicate the predicted miR-101 and miR-494 seed sequences (in bold) using the numbering of the human CFTR 3’UTR (3HSAR032708 from UTRdb database). (B) Predicted CFTR 3’UTR hybrid structure with miR-101 or miR-494 and mean free energy (mfe) obtained by RNAHybrid server.

Figure 2. Mir-101 and mir-494 target the CFTR 3’-UTR.

(A) Schematic representation of the construct used in the luciferase assays. A fragment of 741 bp of the CFTR 3’-UTR, encompassing the putative responsive elements for miR-101 and miR-494, was cloned in pRLTK vector downstream to the Renilla luciferase coding sequence. (B) HEK293 cells were transfected independently with pRLTK control plasmid or pCFTR-3’UTR vector together with either specific microRNA (miR-101 or miR-494) or a negative control miRNA (miR-Ctr). At 48 hour post-transfection, luciferase activity was measured and normalized to the Firefly control. Data are presented as the normalized activity of the indicated miR-transfected cells relative to the negative mimic control (miR-Ctr). These results represent the mean of at least three independent experiments ± standard error (SE), each carried out in triplicate. The significance levels were obtained by ANOVA: **, p<0.01; ***, p<0.001 compared with the control miRNA. (C) Combined miR-101/miR-494 synthetic mimics (100 nM miR-101 and 100 nM miR-494) or 200 nM miR-Ctr (^§) were delivered into HEK293 cells together with pCFTR-3’UTR plasmid. Luciferase activity measured and data reported as in B. Statistical significance from ANOVA: ns, not significant; **, p<0.01; ***p<0.001 compared with the control miRNA; miR-101+miR494 versus miR-Ctr either at 100 nM or at 200 nM.

Figure 3. Specificity of miR-101 and miR-494 CFTR suppression by recognition of the seed sequence.

(A) Schematic representation of the reporter constructs carrying CFTR 3’UTR mutated at miR-101 responsive element or mir-494 seed sequence. (B) Relative luciferase activity in HEK293 cells over-expressing the indicated miRNAs and transfected with the CFTR wild-type 3’UTR vector or its mutant derivative pCFTR-3’UTR-mut101 and pCFTR-3’UTR-mut494. Data are presented as the normalized activity of miR-transfected cells (miR-101 or miR-494) relative to cells transfected with miR-Ctr. All data are average values ± SE from at least three experiments (ANOVA, ns, not significant; *, p<0.05; ***, p<0.001 compared to the basal value with miR-Ctr).