Dataset Information

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.

SUBMITTER: Gillen AE

PROVIDER: S-EPMC4323381 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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

Gillen Austin E AE Gosalia Nehal N Leir Shih-Hsing SH Harris Ann A

The Biochemical journal 20110801 1

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 express ...[more]

PMID: 21689072

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Project description:Background: Most cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that lead to protein misfolding and degradation by the ubiquitin-proteasome system. Previous studies demonstrated that PIAS4 facilitates the modification of wild-type (WT) and F508del CFTR by small ubiquitin-like modifier (SUMO)-1, enhancing CFTR biogenesis by slowing immature CFTR degradation and producing increased immature CFTR band B. Methods: We evaluated two correction strategies using misfolding mutants, including the common variant, F508del. We examined the effects on mutant expression of co-expression with PIAS4 (E3 SUMO ligase), and/or the corrector, C18. To study the impact of these correction conditions, we transfected CFBE410- cells, a bronchial epithelial cell line, with a CFTR mutant plus: (1) empty vector, (2) empty vector plus overnight 5 μM C18, (3) PIAS4, and (4) PIAS4 plus C18. We assessed expression at steady state by immunoblot of CFTR band B, and if present, band C, and the corresponding C:B band ratio. The large PIAS4-induced increase in band B expression allowed us to ask whether C18 could act on the now abundant immature protein to enhance correction above the control level, as reported by the C:B ratio. Results: The data fell into three mutant CFTR categories as follows: (1) intransigent: no observable band C under any condition (i.e., C:B = 0); (2) throughput responsive: a C:B ratio less than control, but suggesting that the increased band C resulted from PIAS4-induced increases in band B production; and (3) folding responsive: a C:B ratio greater than control, reflecting C18-induced folding greater than that expected from increased throughput due to the PIAS4-induced band B level. Conclusion: These results suggest that the immature forms of CFTR folding intermediates occupy different loci within the energetic/kinetic folding landscape of CFTR. The evaluation of their properties could assist in the development of correctors that can target the more difficult-to-fold mutant conformations that occupy different sites within the CFTR folding pathway.

Dataset Information

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

Publications

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

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