miR-16 rescues F508del-CFTR function in native cystic fibrosis epithelial cells

Abstract

Cystic fibrosis (CF) is due to mutations in the CFTR gene, which prevents correct folding, trafficking and function of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) protein. The dysfunctional effect of CFTR mutations, principally the F508del-CFTR mutant, is further manifested by hypersecretion of the pro-inflammatory chemokine interleukin-8 into the airway lumen, which further contributes to morbidity and mortality. We have hypothesized that microRNA (miR)-based therapeutics could rescue the dysfunctional consequences of mutant CFTR. Here we report that a miR-16 mimic can effectively rescue F508del-CFTR protein function in airway cell lines and primary cultures, of differentiated human bronchial epithelia from F508del homozygotes, which express mutant CFTR endogenously. We also identify two other miRs, miR-1 and miR-302a, which are also active. Although miR-16 is expressed at basal comparable levels in CF and control cells, miR-1 and miR-302a are undetectable. When miR mimics are expressed in CF lung or pancreatic cells, the expression of the F508del-CFTR protein is significantly increased. Importantly, miR-16 promotes functional rescue of the cyclic AMP-activated apical F508del-CFTR chloride channel in primary lung epithelial cells from CF patients. We interpret these findings to suggest that these miRs may constitute novel targets for CF therapy.

Figure 1

MicroRNAs (miR) induce F508del-CFTR expression. (a) CFPAC6.0 cells (2 × 10⁵) were transfected with miR mimics, miR-1 (20 nM), miR-16 (25 nM) or miR-302a (30 nM) and pre-miR-negative control (miR-Ctrl) for 48 h with siPort transfection reagent. The cells were lysed and the isolated protein was analyzed using western blot analysis. CFPAC4.7 and native CFPAC6.0 cells were included as controls. The relative quantitation of the C bands compared with total C+B bands are also indicated. The lane with the WT-CFTR protein is part of the same gel. The data are representative of three or more independent experiments. (b) For analyses of CFTR mRNA, CFPAC6.0 cells (2 × 10⁵) were treated as described before, and total RNA was isolated and analyzed using quantitative real-time PCR (qRT-PCR) specific for CFTR mRNA. The comparison of control versus each of the three miRs is indicated (solid lines, *P<0.05). In addition, comparison of miR-16 with miR-1 or miR-302a is also included (dotted lines, *P<0.05). (c) The stability of CFTR mRNA was also measured in native CFPAC6.0 and miR-treated CFPAC6.0 cells. Subsequent to transfection, the RNA was isolated after treatment with actinomycin D (5 μgml ^{− 1}) for the indicated time intervals (0, 1, 2 and 4 h). The remaining RNA was analyzed using qRT-PCR. The data are representative of three or more independent experiments.

Figure 2

Expression and localization of CFTR. (a–a″) CFPAC6.0 cells containing the F508del mutation show low levels of CFTR labeling (green) that is faintly perinuclear and punctate. (b–b″) CFPAC4.7 cells that express WT-CFTR at the endogenous locus show robust, punctate CFTR labeling (green, b, b″) throughout the cytoplasm that can overlap with the cell membrane (b′, b″, red). (c–e″) Treating (ΔF508) CFPAC6.0 cells with miR-16 (c–c″), miR-1 (d–d″) or miR-302a (e–e″) cause a large increase in the amount of CFTR labeling (green), with miR-16 causing the greatest increase. Green=a-CFTR (a, a″, b, b″, c, c″, d, d″, e, e″), red=phalloidin (a′, a″, b′, b″, c′, c″, d′, d″, e′, e′), blue=4’,6-diamidino-2-phenylindole (a–e″). The image depicts Z-stacks, and an average of 300 cells in 10 different fields was analyzed.

Figure 3

FACS analyses of CFTR expression. The increased cell surface expression of CFTR induced by overexpression of miRs was analyzed using flow cytometry in two different CF epithelial cells. (a) IB3-1 CF lung epithelial cells (2 × 10⁵) were transfected with miR mimics, and CFTR expression was compared with that in IB3-1 mock-treated cells and and IB3-1/S9 control cells. (b) Similarly, CFPAC6.0 pancreatic epithelial cells (2 × 10⁵) were transfected with miR mimics, and CFTR expression was compared with that in native CFPAC6.0-mock-treated cells and CFPAC4.7 control cells. The data are representative of three or more independent experiments.

Figure 4

Suppression of IL-8 expression by rescue-competent miRs in CF lung epithelial cells. In silico analyses indicate that both miR-16 and miR-302a have binding sites at the target IL-8 mRNA 3′-UTR sequences. IB3-1 CF lung epithelial cells (2 × 10⁵) were transfected with miR mimics, (a) miR-16 (25 nM) or (b) miR-302a (30 nM) for 48 h with siPort transfection reagent. The RNA was isolated after treatment with actinomycin D (5 μgml ^{− 1}) for the indicated time intervals (0, 1, 2 and 3 h). The remaining IL-8 mRNA was analyzed by qRT-PCR (*P<0.05). The corresponding secreted IL-8 protein was measured using ELISA (*P<0.05). (c) Luciferase reporter assays were performed in IB3-1 CF cells transfected with pMIR-report vectors, either controls or those containing IL-8 3′-UTR target sequences of miR-16 (both WT and mutant), in the presence of pre-miR-16 or control pre-miR. The data reflect averages of at least three independent experiments (*P<0.05 and **P>0.05).

Figure 5

Suppression of HSP90 expression by miR-16 in CF epithelial cells. CFPAC6.0 pancreatic epithelial cells (2 × 10⁵) were transfected with miR-16 mimic as previously described. To determine the effect of miR-16 overexpression on the expression of HSP90, both (a) Hsp90 mRNA (qPCR) and (b) Hsp90 protein (western blot) levels were analyzed (*P<0.05). The two lanes are part of the same gel. The protein data shown here are representative of three or more independent experiments.

Figure 6

Rescue of ΔF508-CFTR function and attenuation of inflammation. CF HBE cells transfected with microRNA (miR) mimics, miR-16 (25 nM) and miR-302a (30 nM) for 48 h in air–liquid interface cultures. The expression levels of (a) miR-16 and (b) miR-302a in the CF HBE cells were analyzed using miR-specific Taqman assays. (c) The cAMP-activated short-circuit current was measured after administration of forskolin (10 μM)+P2 (50 μM; *P<0.05). (d) Subsequently, these cells were treated with CFTRinh-172 to inhibit CFTR, followed by measurement of short-circuit current (*P<0.05). The CF HBE cells were lysed and total RNA was isolated and analyzed using the Taqman assays for (e) IL-8 mRNA and (f) miR-155 (*P<0.05).