Identification of a novel functional miR-143-5p recognition element in the Cystic Fibrosis Transmembrane Conductance Regulator 3’UTR

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs involved in regulation of gene expression. They bind in a sequence-specific manner to miRNA recognition elements (MREs) located in the 3″ untranslated region (UTR) of target mRNAs and prevent mRNA translation. MiRNA expression is dysregulated in cystic fibrosis (CF), affecting several biological processes including ion conductance in the epithelial cells of the lung. We previously reported that miR-143 is up-regulated in CF bronchial brushings compared to non-CF. Here we identified two predicted binding sites for miR-143-5p (starting at residues 558 and 644) on the CFTR mRNA, and aimed to assess whether CFTR is a true molecular target of miR-143-5p. Expression of miR-143-5p was found to be up-regulated in a panel of CF vs non-CF cell lines (1.7-fold, P = 0.0165), and its levels were increased in vitro after 20 hours treatment with bronchoalveolar lavage fluid from CF patients compared to vehicle-treated cells (3.3-fold, P = 0.0319). Luciferase assays were performed to elucidate direct miRNA::target interactions and showed that miR-143-5p significantly decreased the reporter activity when carrying the wild-type full length sequence of CFTR 3″UTR (minus 15%, P = 0.005). This repression was rescued by the disruption of the first, but not the second, predicted MRE, suggesting that the residue starting at 558 was the actual active binding site. In conclusion, we here showed that miR-143-5p modestly but significantly inhibits CFTR, improving the knowledge on functional MREs within the CFTR 3″UTR. This could lead to the development of novel therapeutic strategies where miRNA-mediated CFTR repression is blocked thereby possibly increasing the efficacy of the currently available CFTR modulators.

Keywords

microRNA - cystic fibrosis - CFTR - qRT-PCR - luciferase assay

Publication History

Received: 01 September 2017

Accepted: 08 February 2018

Article published online:
10 May 2021

• References

• 1 Lee RC, Ambros V. An extensive class of small RNAs in Caenorhabditis elegans. Science 1993; 294: 862-864
• 2 Chandra S, Vimal D, Sharma D. et al. Role of miRNAs in development and disease: Lessons learnt from small organisms. Life Sci 2017; 185: 8-14
• 3 Rupaimoole R, Calin GA, Lopez-Berestein G. et al. miRNA Deregulation in Cancer Cells and the Tumor Microenvironment. Cancer Discov 2016; 6: 235-246
• 4 Szymczak I, Wieczfinska J, Pawliczak R. Molecular Background of miRNA Role in Asthma and COPD: An Updated Insight. Biomed Res Int 2016; 2016: 7802521
• 5 Chen JQ, Papp G, Szodoray P. The role of microRNAs in the pathogenesis of autoimmune diseases. Autoimmun Rev 2016; 15: 1171-1180
• 6 Kiriakidou M, Nelson PT, Kouranov A. et al. A combined computational-experimental approach predicts human microRNA targets. Genes Dev 2004; 18: 1165-1178
• 7 Lai EC, Tam B, Rubin GM. Pervasive regulation of Drosophila Notch target genes by GY-box-, Brd-box-, and K-box-class microRNAs. Genes Dev 2005; 19: 1067-1080
• 8 Vodicka P, Pardini B, Vymetalkova V. et al. Polymorphisms in Non-coding RNA Genes and Their Targets Sites as Risk Factors of Sporadic Colorectal Cancer. Adv Exp Med Biol 2016; 937: 123-149
• 9 Nossent AY, Hansen JL, Doggen C. et al. SNPs in microRNA binding sites in 3″-UTRs of RAAS genes influence arterial blood pressure and risk of myocardial infarction. Am J Hypertens 2011; 24: 999-1006
• 10 Wang X, Jiang H, Wu W. et al. An Integrating Approach for Genome-Wide Screening of MicroRNA Polymorphisms Mediated Drug Response Alterations. Int J Genomics 2017; 2017: 1674827
• 11 Thomson DW, Bracken CP, Goodall GJ. Experimental strategies for microRNA target identification. Nucleic Acids Res 2011; 39: 6845-6853
• 12 Gillen AE, Gosalia N, Leir SH. et al. MicroRNA regulation of expression of the cystic fibrosis transmembrane conductance regulator gene. Biochem J 2011; 438: 25-32
• 13 Megiorni F, Cialfi S, Dominici C. et al. Synergistic post-transcriptional regulation of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) by miR-101 and miR-494 specific binding. PLoS One 2011; 6: e26601
• 14 Hassan F, Nuovo GJ, Crawford M. et al. MiR-101 and miR-144 regulate the expression of the CFTR chloride channel in the lung. PLoS One 2012; 7: e50837
• 15 Oglesby IK, Chotirmall SH, McElvaney NG. et al. Regulation of cystic fibrosis transmembrane conductance regulator by microRNA-145, -223, and -494 is altered in ⍼F508 cystic fibrosis airway epithelium. J Immunol 2013; 190: 3354-3362
• 16 Amato F, Seia M, Giordano S. et al. Gene mutation in microRNA target sites of CFTR gene: a novel pathogenetic mechanism in cystic fibrosis?. PLoS One 2013; 8: e60448
• 17 Ramachandran S, Karp PH, Osterhaus SR. et al. Post-transcriptional regulation of cystic fibrosis transmembrane conductance regulator expression and function by microRNAs. Am J Respir Cell Mol Biol 2013; 49: 544-551
• 18 Viart V, Bergougnoux A, Bonini J. et al. Transcription factors and miRNAs that regulate fetal to adult CFTR expression change are new targets for cystic fibrosis. Eur Respir J 2015; 45: 116-128
• 19 World Health Organization. The molecular genetic epidemiology of cystic fibrosis: report of a joint meeting of WHO/ECFTN/ICF(M)A/ECFS; June 19, 2002; Genoa, Italy
• 20 The Clinical and Functional TRanslation of CFTR (CFTR2), 2017. Available from: http://cftr2.org
• 21 McKiernan PJ, Greene CM. MicroRNA Dysregulation in Cystic Fibrosis. Mediators Inflamm 2015; 2015: 529642
• 22 Oglesby IK, Bray IM, Chotirmall SH. et al. miR-126 is downregulated in cystic fibrosis airway epithelial cells and regulates TOM1 expression. J Immunol 2010; 184: 1702-1709
• 23 Oglesby IK, Vencken SF, Agrawal R. et al. miR-17 overexpression in cystic fibrosis airway epithelial cells decreases interleukin-8 production. Eur Respir J 2015; 46: 1350-1360
• 24 MirkoviĆ B, Murray MA, Lavelle GM. et al. The Role of Short-Chain Fatty Acids, Produced by Anaerobic Bacteria, in the Cystic Fibrosis Airway. Am J Respir Crit Care Med 2015; 192: 1314-1324
• 25 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25: 402-408
• 26 Agarwal V, Bell GW, Nam JW. et al. Predicting effective microRNA target sites in mammalian mRNAs. Elife 2015;4
• 27 Betel D, Koppal A, Agius P. et al. Comprehensive modeling of microRNA targets predicts functional non-conserved and non-canonical sites. Genome Biol 2010; 11: R90
• 28 Miranda KC, Huynh T, Tay Y. et al. A pattern-based method for the identification of MicroRNA binding sites and their corresponding heteroduplexes. Cell 2006; 126: 1203-1217
• 29 ClÉment T, Salone V, Rederstorff M. Dual luciferase gene reporter assays to study miRNA function. Methods Mol Biol 2015; 1296: 187-198
• 30 Karimi L, Mansoori B, Shanebandi D. et al. Function of microRNA-143 in different signal pathways in cancer: New insights into cancer therapy. Biomed Pharmacother 2017; 91: 121-131
• 31 He M, Zhan M, Chen W. et al. MiR-143-5p Deficiency Triggers EMT and Metastasis by Targeting HIF-1⤙ in Gallbladder Cancer. Cell Physiol Biochem 2017; 42: 2078-2092
• 32 Wu XL, Cheng B, Li PY. et al. MicroRNA-143 suppresses gastric cancer cell growth and induces apoptosis by targeting COX-2. World J Gastroenterol 2013; 19: 7758-7765
• 33 FDA expands approved use of Kalydeco to treat additional mutations of cystic fibrosis, FDA News Release. 2017 Available from: https://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm559212.htm
• 34 Fajac I, De Boeck K. New horizons for cystic fibrosis treatment. Pharmacol Ther 2017; 170: 205-211
• 35 Clancy JP, Rowe SM, Accurso FJ. et al. Results of a phase IIa study of VX-809, an investigational CFTR corrector compound, in subjects with cystic fibrosis homozygous for the F508del-CFTR mutation. Thorax 2012; 67: 12-18
• 36 Boyle MP, Bell SC, Konstan MW. et al. A CFTR corrector (lumacaftor) and a CFTR potentiator (ivacaftor) for treatment of patients with cystic fibrosis who have a phe508del CFTR mutation: a phase 2 randomised controlled trial. Lancet Respir Med 2014; 2: 527-538
• 37 Wainwright CE, Elborn JS, Ramsey BW. et al. Lumacaftor-Ivacaftor in Patients with Cystic Fibrosis Homozygous for Phe508del CFTR. N Engl J Med 2015; 373: 220-231
• 38 Zarrilli F, Amato F, Morgillo CM. et al. Peptide Nucleic Acids as miRNA Target Protectors for the Treatment of Cystic Fibrosis. Molecules 2017; 22: 1144
• 39 Cantin AM. Cystic Fibrosis Transmembrane Conductance Regulator. Implications in Cystic Fibrosis and Chronic Obstructive Pulmonary Disease. Ann Am Thorac Soc 2016; 13 Suppl. 2: S150-S155
• 40 Solomon GM, Raju SV, Dransfield MT. et al. Therapeutic Approaches to Acquired Cystic Fibrosis Transmembrane Conductance Regulator Dysfunction in Chronic Bronchitis. Ann Am Thorac Soc 2016; 13 Suppl. 2: S169-S176