A microRNA network regulates expression and biosynthesis of wild-type and DeltaF508 mutant cystic fibrosis transmembrane conductance regulator

Abstract

Production of functional proteins requires multiple steps, including gene transcription and posttranslational processing. MicroRNAs (miRNAs) can regulate individual stages of these processes. Despite the importance of the cystic fibrosis transmembrane conductance regulator (CFTR) channel for epithelial anion transport, how its expression is regulated remains uncertain. We discovered that miRNA-138 regulates CFTR expression through its interactions with the transcriptional regulatory protein SIN3A. Treating airway epithelia with an miR-138 mimic increased CFTR mRNA and also enhanced CFTR abundance and transepithelial Cl(-) permeability independent of elevated mRNA levels. An miR-138 anti-miR had the opposite effects. Importantly, miR-138 altered the expression of many genes encoding proteins that associate with CFTR and may influence its biosynthesis. The most common CFTR mutation, ΔF508, causes protein misfolding, protein degradation, and cystic fibrosis. Remarkably, manipulating the miR-138 regulatory network also improved biosynthesis of CFTR-ΔF508 and restored Cl(-) transport to cystic fibrosis airway epithelia. This miRNA-regulated network directs gene expression from the chromosome to the cell membrane, indicating that an individual miRNA can control a cellular process more broadly than recognized previously. This discovery also provides therapeutic avenues for restoring CFTR function to cells affected by the most common cystic fibrosis mutation.

Fig. 1.

miR-138 and SIN3A regulate CFTR expression in airway epithelia. (A) SIN3A mRNA abundance in human primary airway epithelia at 24 h after the indicated interventions (n = 6). Scr, negative control; SIN3A DsiRNA, positive control; UnT, untransfected cells. (B) SIN3A protein abundance in primary airway epithelia at 72 h posttransfection. A representative immunoblot is shown. (C) CFTR mRNA abundance in Calu-3 cells at 24 h after indicated transfections. CFTR DsiRNA, positive control. (D) CFTR protein abundance in Calu-3 cells at 72 h posttransfection (R-769 antibody). (E and F) Changes in conductance (G_t) (E) and transepithelial current (I_t) (F) with indicated treatments. Basal resistance range, 397–586 ohm*cm². Error bars indicate mean ± SE. *P < 0.01 relative to Scr; ⁺P < 0.01, ⁺⁺P < 0.01 relative to ΔG_t and ΔI_t in Scr-transfected samples on forskolin and IBMX (F&I) and CFTR inhibitor GlyH-101 treatment, respectively.

Fig. 2.

miR-138 and SIN3A regulate CFTR expression in primary cultures of human airway epithelia and cells with no CFTR expression. (A) CFTR mRNA abundance in primary airway epithelia at 24 h after interventions (n = 6). (B) CFTR protein abundance from primary airway epithelia at 72 h posttransfection; R-769 antibody, representative immunoblot. (C and D) Changes in conductance (G_t) (C) and transepithelial current (I_t) (D) with indicated treatments. Each bar represents six primary airway epithelial cell cultures each from three donors, pretransfected with the indicated reagents. Basal resistance range, 415–672 ohm*cm². (E) CFTR protein abundance in HeLa cells; R-769 antibody. (F) Schematic representing miR-138– and SIN3A-mediated regulation of CFTR expression. (G) Fold enrichment of SIN3A, assessed by quantitative PCR after ChIP. Data are normalized to CFTR intron 17a DHS. (Inset) CTCF immunoblot of lysates from three airway epithelia donors. Error bars indicate mean ± SE; *P < 0.01 relative to Scr; **P < 0.01 relative to intron 17a; ⁺P < 0.01 and ⁺⁺P < 0.01 relative to ΔG_t and ΔI_t in Scr-transfected samples on F&I and GlyH-101 treatment, respectively.

Fig. 3.

miR-138 regulates CFTR processing. (A) Surface display, as detected by ELISA, of epitope-tagged CFTR in CFTR-3HA HeLa cells transfected with indicated reagents. (B) CFTR protein abundance in CFTR-3HA HeLa cells at 24 h posttransfection. (Upper) Anti-HA antibody. (Lower) R769 antibody. (C) Schematic showing regions of intersection of SIN3A DsiRNA, miRNA-mimic, and CFTR-associated genes data sets; P < 0.05 (SI Appendix, Tables S2–S4). (D) Surface display of epitope-tagged CFTR in CFTR-ΔF508-3HA HeLa cells transfected with indicated reagents. (E) CFTR protein abundance in CFTR-ΔF508-3HA HeLa cells at 24 h posttransfection. (Upper) Anti-HA antibody. (Lower) R769 antibody. Error bars indicate mean ± SE; *P < 0.01 relative to Scr.

Fig. 4.

SIN3A inhibition yields partial rescue of Cl⁻ transport in CF epithelia. (A) (Upper) CFTR protein abundance from airway epithelia (CFTR Q493X/S912X, 24-1 antibody) after indicated treatments. (Lower) Change in I_t values after F&I stimulation and GyH-101 inhibition (one donor, three replicates). Basal resistance range, 279–360 ohm*cm². (B) Representative CFTR immunoblot from primary epithelia (CFTR ΔF508/ΔF508) at 72 h posttransfection; R-769 antibody, donor 2 in D. (C) Responses of CFTR ΔF508/ΔF508 epithelia to indicated interventions (donor 1). (Upper) I_t tracings of responses to F&I, followed by GlyH-101 treatment (epithelia pretreated with amiloride and DIDS). (Lower) Summary of change in I_t in response to F&I, followed by GlyH-101 treatment (one donor, eight replicates). Basal resistance range, 488–691 ohm*cm². Error bars indicate mean ± SE. *P < 0.01, **P < 0.01 relative to ΔI_t in Scr-transfected samples after F&I and GlyH-101 treatments, respectively; ⁺P < 0.01 relative to Scr. (D) Changes in I_t values after F&I treatment of six primary CF airway epithelia cultures transfected with indicated reagents. Six untreated or Scr-treated CF samples served as negative controls; eight non-CF samples served as WT controls. ΔF/* denotes ΔF508/3659delC; ΔF/** denotes ΔF508/R1162X. Horizontal bars indicate means. Basal resistance range, 295–819 ohm*cm². (E) Working model of steps in CFTR transcription and protein biosynthesis pathway in which miR-138–regulated gene products influence WT and CFTR-ΔF508 (Fig. 3C and SI Appendix, Tables S2–S5).