Association of MicroRNA-196a2 Variant with Response to Short-Acting β2-Agonist in COPD: An Egyptian Pilot Study

Abstract

Chronic obstructive pulmonary disease (COPD) is a multifactorial chronic respiratory disease, characterized by an obstructive pattern. Understanding the genetic predisposition of COPD is essential to develop personalized treatment regimens. MicroRNAs (miRNAs) are small, endogenous, non-coding RNAs that modulate the expression levels of specific proteins based on sequence complementarity with their target mRNA molecules. Emerging evidences demonstrated the potential use of miRNAs as a disease biomarker. This pilot study aimed to investigate the association of the MIR-196a2 rs11614913 (C/T) polymorphism with COPD susceptibility, the clinical outcome and bronchodilator response to short-acting β2-agonist. Genotyping of rs11614913 polymorphism was determined in 108 COPD male patients and 116 unrelated controls using real-time polymerase chain reaction technology. In silico target prediction and network core analysis were performed. COPD patients did not show significant differences in the genotype distribution (p = 0.415) and allele frequencies (p = 0.306) of the studied miRNA when compared with controls. There were also no associations with GOLD stage, dyspnea grade, disease exacerbations, COPD assessment test for estimating impact on health status score, or the frequency of intensive care unit admission. However, COPD patients with CC genotype corresponded to the smallest bronchodilator response after Salbutamol inhalation, the heterozygotes (CT) had an intermediate response, while those with the TT genotype showed the highest response (p < 0.001). In conclusion MIR-196a2 rs11614913 polymorphism is associated with the bronchodilator response of COPD in our sample of the Egyptian population, generating hypothesis of the potential use of MIR-196a2 variant as a pharmacogenetic marker for COPD.

Fig 1. Workflow of in silico data analysis.

Gene and microRNA sequence and structure were retrieved from miRBase database (http://microrna.sanger.ac.uk/targets/v3/). Multiple computational prediction tools were employed to identify miR-196a2 target genes (in 3'URT, 5'UTR, and CDS) as miRDB, miRNAMap, TargetScanHuman v6.2, miRTarBase v18 and DIANA-microT-CDS v5.0 databases. Result intersection, statistical validation, and filtration of the putative miRNA targets were applied to reduce the false positive prediction rate. The predicted miRNA target genes were analyzed for gene ontology (GO) terms and KEGG enrichment pathway analysis using DIANA-miRPath v2.0 web-server (http://diana.imis.athenainnovation.gr/DianaTools/index.php?r=mirpath/index) and miRTar Human tool (http://miRTar.mbc.nctu.edu.tw/). MiRNA-196a2-disease association was explored using a miRPub server (http://www.microrna.gr/mirpub/). Gene variations and frequencies in various populations were obtained from Ensembl (http://www.ensembl.org/) and miRdSNP databases (http://mirdsnp.ccr.buffalo.edu/). The Impact of the SNP on secondary structure was predicted based on the minimum free energy using miRNAMap 2.0 and RNAfold server. Comparative functional analysis between predicted target gene sets in wild and mutant variants were performed using miRmut2Go. SNP identification in the study groups aimed to assess its association with disease risk, severity and pulmonary function.

Fig 2. Mapping MIR196A2 gene variations.

(a) Human hsa-miR-196a2 gene has 2 mature miRNA variants located within the sequence of the mature miRNA and 6 non-coding transcript variants. Chromosomal location and coordinates in base pair are derived from human genome assembly GRCh38. Eight gene variations were retrieved from Ensembl. Variant ID, alternative nucleotides, and minor allele frequency (MAF) are shown. All polymorphisms were very rare (MAF<0.1), except the studied variant. (b) Mature miR and miR* sequences are underlined. Green highlighted nucleotides are non-coding transcript variants in pre-miR-196a2. Yellow highlighted nucleotides are mature miRNA variants at 5p arm. Red arrow indicates the studied common variant.

Fig 3. MicroRNA-196a2 predicted target gene products with putative roles in chronic obstructive pulmonary disease.

The diagram was manually curated by combining in silico analysis of predicted hsa-miR-196a2-targeted signaling pathways involved in the pathogenesis of COPD and review of previous literatures. KEGG pathways (blue); predicted target genes of miR-196a2 (yellow box); other genes in the pathway network (white box); activate (black arrow); inhibit (red line). TGFB transforming growth factor beta, ACVR2B activin A receptor type IIB, SMAD2/3, human mothers against decapentaplegic homolog 2/3, ECM extracellular matrix, TGFBR2 TGFB receptor 2, ROCK1 rho-associated coiled-coil containing protein kinase 1, MLC myosin light chain, DIAPH2 diaphanous homolog 2, OCRL oculocerebrorenal syndrome of Lowe, COL3A1 collagen type 3 alpha 1, ITGB integrin beta, ITGAV integrin alpha V, FAK focal adhesion kinase, EPB4L2 erythrocyte membrane protein band 4-like 2, GF growth factor; PDGFRA platelet-derived growth factor receptor alpha polypeptide, GRB2 growth factor receptor binding protein 2, mSOS mammalian son of sevenless, RAS rat associated sarcoma, MEK mitogen-activated protein kinase and extracellular regulated kinase kinase, c-myc cellular oncogene originally identified as the transforming determinant of avian myelocytomatosis virus, c-Fos cellular oncogene homologue to that of FINKEL induce murine osteosarcoma, MAPK mitogen-activated protein kinase, c-Jun cellular Proto-oncogene protein Jun, cycD cyclin D, TLR Toll like receptor, TAK TGF-beta activated kinase, TNFa, Tumor necrosis factor alpha, IL-6 interleukin-6.

Fig 4. Predicted functional impact of rs11614913 pre-miR-196a2 variant.

Hsa-miR-196a-2 is composed of two different mature miRNAs (miR-196a and miR196a*), which are processed from the same stem-loop. The SNP (rs11614913) lies in the mature sequence of miR-196a* but may influence either miRNA by affecting processing of the pre-miRNA to its mature form [47].