miR-200b downregulates CFTR during hypoxia in human lung epithelial cells

Abstract

Background: Hypoxic conditions induce the expression of hypoxia-inducible factors (HIFs) that allow cells to adapt to the changing conditions and alter the expression of a number of genes including the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a low abundance mRNA in airway epithelial cells even during normoxic conditions, but during hypoxia its mRNA expression decreases even further.

Methods: In the current studies, we examined the kinetics of hypoxia-induced changes in CFTR mRNA and protein levels in two human airway epithelial cell lines, Calu-3 and 16HBE14o-, and in normal primary bronchial epithelial cells. Our goal was to examine the posttranscriptional modifications that affected CFTR expression during hypoxia. We utilized in silico predictive protocols to establish potential miRNAs that could potentially regulate CFTR message stability and identified miR-200b as a candidate molecule.

Results: Analysis of each of the epithelial cell types during prolonged hypoxia revealed that CFTR expression decreased after 12 h during a time when miR-200b was continuously upregulated. Furthermore, manipulation of the miRNA levels during normoxia and hypoxia using miR-200b mimics and antagomirs decreased and increased CFTR mRNA levels, respectively, and thus established that miR-200b downregulates CFTR message levels during hypoxic conditions.

Conclusion: The data suggest that miR-200b may be a suitable target for modulating CFTR levels in vivo.

Keywords: CFTR; HIF-1; hsa-miR-200b-3p; micro-RNA 200b.

Fig. 1

Regulation of CFTR during hypoxia in human lung epithelial cells, Calu3 cells (left panels) and 16HBE14o- cells (right panels). a CFTR mRNA is reduced during hypoxia. CFTR mRNA levels were monitored in qRT-PCR experiments. The results from 3 independent experiments (n = 12) are plotted normalized to 18S rRNA levels and expressed as a fold-change over the normoxic control. b Hypoxia sequentially increases HIF-1α protein levels and reduces CFTR protein levels. Protein expression levels of were monitored with SDS-PAGE and Western Blot and normalized to β-actin levels. Two individual samples (4 μg of total protein per lane) were tested for each time point and the experiments were repeated twice. Error bars represent standard deviations. Significant changes (P < 0.05) are marked with an asterisk

Fig. 2

Downregulation of CFTR expression during hypoxia is HIF-1 dependent and relies on both the 5′ and 3′ UTRs of CFTR mRNA. a Calu3, 16HBE14o- and HeLa WT CFTR cells were treated with hypoxia mimetics (500 μM DMOG for 12 h (light grey) and 200 μM CoCl₂ for 12 h (dark grey)) and the mRNA levels were monitored in qRT-PCR experiments. CFTR mRNA levels from 2 independent experiments (n = 8) are plotted relative to 18S rRNA levels and expressed as a fold change over the untreated control. b Calu3 cells were transfected with 5′UTR CFTR luciferase reporter (white), 3′UTR CFTR luciferase reporter (light grey) and co-transfected with both 5′UTR and 3′UTR CFTR luciferase reporters (dark grey) and treated with hypoxia mimetics (500 μM DMOG or 200 μM CoCl₂ for 12 h) and the luciferase activity was monitored. These reporters were normalized to internal controls (Renilla) firefly luciferase activities from 2 independent experiments (n = 6) and plotted and expressed as a fold-change over non-treated control. Error bars represent standard deviations. Significant changes (P < 0.05) are marked with an asterisk

Fig. 3

Hypoxia induces miR-200b in human airway epithelial cells in a HIF-1-dependent manner. a The predicted target site of miR-200b in CFTR 3′UTR is shown above. The miR-200b target site was predicted in human CFTR 3′UTR only, Hypoxia-induced changes in the expression profiles of miR-200b and miR-200c in Calu3 and 16HBE14o- cells are shown. The miRNA levels were monitored in qRT-PCR experiments. The results from 2 independent experiments (n = 8) are plotted normalized to RNU48 and expressed as a fold-change over the normoxic control. b Calu3 and 16HBE14o- cells were treated with hypoxia mimetic (200 μM CoCl₂ for 12 h) and the miRNA levels were monitored in qRT-PCR experiments. miR-200b and miR-200c levels were measured in 3 independent experiments (n = 10) and are plotted relative to RNU44 levels and expressed as a fold change over the untreated controls. Error bars represent standard deviations (SD). Significant changes (P < 0.05) are marked with an asterisk

Fig. 4

miR-200b binds to the predicted target sequence in the CFTR 3′UTR. a HEK293 cells were transfected with 3′UTR CFTR luciferase reporter construct alone (white) or together with miR-200b mimic (grey, left panel) or miR-200c mimic (grey, right panel). Similar experiments were performed on control vector that did not contain the miR-200b/miR-200c target site (not shown). Data were normalized to control Renilla luciferase activities from 2 independent experiments (n = 6) and are expressed as a fold-change over control. Error bars represent standard deviations (SD). Significant changes (P < 0.05) are marked with an asterisk. b The comparison of miR-200b and miR-200c seed sequences is shown

Fig. 5

miR-200b decreases the expression of CFTR mRNA during normoxia and hypoxia. a Calu3 and 16HBE14o- cells were transfected with miR-200b mimic or antagomir and the mRNA levels were monitored in qRT-PCR experiments in normoxic conditions and after 12 h of hypoxia. CFTR mRNA levels from 2 independent experiments (n = 8) are normalized to 18S rRNA levels and expressed as a fold change over the transfection control. b The corresponding changes in CFTR protein levels of were detected with SDS-PAGE and Western Blot analyses and normalized to the β-actin levels. Two individual samples (3 μg of total protein per lane) were tested for each treatment and the experiments were repeated twice. Error bars represent standard deviations (SD). Significant changes (P < 0.05) are marked with an asterisk

Fig. 6

miR-200b decreases the expression of CFTR mRNA during normoxia and hypoxia in primary normal human bronchial epithelial cells (NHBEC). a The levels of miR-200b after 12 h of hypoxia in NHBECs from 3 independent experiments (n = 6) are plotted normalized to RNU44 levels and expressed as a fold change over the normoxia control. b NHBEC cells were transfected with miR-200b mimic or antagomir and the mRNA levels were monitored in qRT-PCR experiments in normoxic conditions and after 12 h of hypoxia. CFTR mRNA levels from 2 independent experiments (n = 8) are plotted normalized to 18S rRNA levels and expressed as a fold change over the transfection control. c The corresponding changes of CFTR protein levels were monitored with SDS-PAGE and Western Blot analysis and normalized to the β-actin levels. Two individual samples (3 μg of total protein per lane) were tested for each treatment and the experiments were repeated twice. Error bars represent standard deviations (SD). Significant changes (P < 0.05) are marked with an asterisk

Fig. 7

Model for negative regulation of CFTR expression during hypoxia by HIF-1 and miR-200b. During hypoxia, HIF-1 activity is induced and HIF-1 binds to the hypoxia response element (HRE) sequence located in CFTR 5′UTR and has been reported to decrease CFTR expression (Zheng et al. [18]). Our studies show that HIF-1 induces miR-200b expression and binds to the target sequence (TS miR-200b) located at the 3′UTR of CFTR mRNA, which further decreases CFTR mRNA and protein expression