MicroRNA regulation of expression of the cystic fibrosis transmembrane conductance regulator gene

Abstract

The CFTR (cystic fibrosis transmembrane conductance regulator) gene shows a complex temporal and spatial pattern of expression that is controlled by multiple cis-acting elements interacting with the basal promoter. Although significant progress has been made towards understanding these genomic elements, there have been no reports of post-transcriptional regulation of CFTR by miRNAs (microRNAs). In the present study, we identify two miRNAs, hsa-miR-145 and hsa-miR-494, which regulate CFTR expression by directly targeting discrete sites in the CFTR 3' UTR (untranslated region). We show that at least 12 miRNAs are capable of repressing endogenous CFTR mRNA expression in the Caco-2 cell line. Ten of these also inhibit expression of a reporter construct containing the CFTR 3' UTR in one or more cell lines, and five repress endogenous CFTR protein expression in Caco-2 cells. Moreover, at least three are expressed in primary human airway epithelial cells, where CFTR expression is maintained at low levels in comparison with intestinal cell lines. Three of the miRNAs that target CFTR, hsa-miR-384, hsa-miR-494 and hsa-miR-1246, also inhibit expression of a reporter carrying the Na(+)-K(+)-Cl(-) co-transporter SLC12A2 [solute carrier family 12 (Na(+)-K(+)-Cl(-) transporters), member 2] 3' UTR, suggesting that these miRNAs may play a more general role in regulating chloride transport in epithelial cells.

Figure 1. The CFTR 3′ UTR and predicted miRNAs

The CFTR 3′ UTR, and the location of predicted miRNA seed sites, mammalian conservation and sequence alignment with relevant mammals. ‘Broadly conserved’ miRNAs are conserved across most vertebrates; ‘conserved’ miRNAs are conserved across most mammals. ‘Placental Mammal Basewise Conservation’ is based on sequence alignment of 33 mammalian genomes, including those appearing individually below [–48]. The Figure is based on the output from http://genome.ucsc.edu (hg19 assembly) [49] and http://www.targetscan.org [25].

Figure 2. Repression of CFTR expression in Caco-2 cells by miRNAs predicted with TargetScan

(A) Expression of CFTR mRNA in Caco-2 cells, measured by TaqMan qRT-PCR, after transient transfection of the indicated miRNA mimics. miRNAs are ordered by TargetScan context score, with the strongest predictions on the left-hand side. (B) ImageJ quantification of CFTR protein expression in Caco-2 cells, measured by Western blot analysis, after transient transfection of the indicated miRNA mimics (average of two biological replicates). *P < 0.05, **P < 0.01 and ***P < 0.001. Values are means ± S.E.M.

Figure 3. miRNA-mediated CFTR repression acts through the 3′ UTR

(A) The pMIR-REPORT-based luciferase reporter constructs used in these assays. (B–D) Luciferase expression from pMIR-CFTR (full-length CFTR 3′ UTR) after co-transfection with pMIR-REPORT β-galactosidase and the indicated miRNA mimics in Caco-2 (B), PANC-1 (C) and 16HBE14o- (D) cells. miRNAs are ordered by TargetScan context score, with the strongest predictions on the left-hand side. (E) Luciferase expression from pMIR-CFTR, pMIR-CFTRmut145 and pMIR-CFTRmut494 after co-transfection with pMIR-REPORT β-galactosidase and the indicated miRNA mimics in Caco-2 cells. *P < 0.05, **P < 0.01 and ***P <0.001. Values are means ± S.E.M. (n ≥ 9).

Figure 4. Expression of miRNA-101, miRNA-145, miRNA-331-3p and miRNA-494 in (A) cell lines and (B) primary human tracheal and bronchial cells (HTEs and NHBEs)

Expression of hsa-miR-101, hsa-miR-145, hsa-miR-331-3p and hsa-miR-494, measured by qRT-PCR with TaqMan miRNA assays. *P < 0.05, **P < 0.01 and ***P < 0.001. Values are means ± S.E.M. Data from three cultures of HTEs and NHBEs are combined to account for variation between individuals.

Figure 5. Repression of SLC12A2 expression by miRNAs that also target CFTR

(A) The SLC12A2 3′ UTR, and the location of predicted miRNA seed sites. The Figure is based on the output from http://genome.ucsc.edu (hg19 assembly) [49] and http://www.targetscan.org [25]. (B–C) Expression of SLC12A2 mRNA in Caco-2 (B) and PANC-1 (C) cells, measured by TaqMan qRT-PCR, after transient transfection of the indicated miRNA mimics. miRNAs are ordered by TargetScan context score, with the strongest predictions on the left-hand side. (D) Luciferase expression from pMIR-SLC12A2 (full-length SLC12A2 3′ UTR) after co-transfection with pMIR-REPORT β-galactosidase and the indicated miRNA mimics in PANC-1 cells. miRNAs are ordered by TargetScan context score, with the strongest predictions on the left-hand side. *P <0.05, **P < 0.01 and ***P < 0.001. Values are means ± S.E.M. (n ≥ 9).