RESUMEN
The long noncoding RNA CDKN2B-AS1 harbors a major coronary artery disease risk haplotype, which is also associated with progressive forms of the oral inflammatory disease periodontitis as well as myocardial infarction (MI). Despite extensive research, there is currently no broad consensus on the function of CDKN2B-AS1 that would explain a common molecular role of this lncRNA in these diseases. Our aim was to investigate the role of CDKN2B-AS1 in gingival cells to better understand the molecular mechanisms underlying the increased risk of progressive periodontitis. We downregulated CDKN2B-AS1 transcript levels in primary gingival fibroblasts with LNA GapmeRs. Following RNA-sequencing, we performed differential expression, gene set enrichment analyses and Western Blotting. Putative causal alleles were searched by analyzing associated DNA sequence variants for changes of predicted transcription factor binding sites. We functionally characterized putative functional alleles using luciferase-reporter and antibody electrophoretic mobility shift assays in gingival fibroblasts and HeLa cells. Of all gene sets analysed, collagen biosynthesis was most significantly upregulated (Padj=9.7 × 10- 5 (AUC > 0.65) with the CAD and MI risk gene COL4A1 showing strongest upregulation of the enriched gene sets (Fold change = 12.13, Padj = 4.9 × 10- 25). The inflammatory "TNFA signaling via NFKB" gene set was downregulated the most (Padj=1 × 10- 5 (AUC = 0.60). On the single gene level, CAPNS2, involved in extracellular matrix organization, was the top upregulated protein coding gene (Fold change = 48.5, P < 9 × 10- 24). The risk variant rs10757278 altered a binding site of the pathogen responsive transcription factor STAT1 (P = 5.8 × 10- 6). rs10757278-G allele reduced STAT1 binding 14.4% and rs10757278-A decreased luciferase activity in gingival fibroblasts 41.2% (P = 0.0056), corresponding with GTEx data. CDKN2B-AS1 represses collagen gene expression in gingival fibroblasts. Dysregulated collagen biosynthesis through allele-specific CDKN2B-AS1 expression in response to inflammatory factors may affect collagen synthesis, and in consequence tissue barrier and atherosclerotic plaque stability.
Asunto(s)
Colágeno , Fibroblastos , Encía , ARN Largo no Codificante , Humanos , ARN Largo no Codificante/genética , Encía/metabolismo , Encía/patología , Fibroblastos/metabolismo , Colágeno/metabolismo , Colágeno/genética , Periodontitis/genética , Periodontitis/metabolismo , Regulación de la Expresión Génica , Células HeLa , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/metabolismo , Enfermedad de la Arteria Coronaria/patología , Infarto del Miocardio/genética , Infarto del Miocardio/metabolismoRESUMEN
AIM: R-spondin 4 (RSPO4) is a suggestive risk gene of stage III-IV, grade C periodontitis and upregulated in gingiva of mice resistant to bacteria-induced alveolar bone loss. We aimed to replicate the association, identify and characterize the putative causal variant(s) and molecular effects, and understand the downstream effects of RSPO4 upregulation. MATERIALS AND METHODS: We performed a two-step association study for RSPO4 with imputed genotypes of a German-Dutch (896 stage III-IV, grade C periodontitis cases, 7104 controls) and Spanish sample (441 cases and 1141 controls). We analysed the allelic effects on transcription factor binding sites with reporter gene and antibody electrophoretic mobility shift assays. We used CRISPR/dCas9 activation and RNA sequencing to pinpoint RSPO4 as the target gene and to analyse downstream effects. RESULTS: RSPO4 was associated with periodontitis (rs6056178, pmeta = 4.6 × 10-5 ). rs6056178 contains a GATA-binding motif. The rs6056178 T-allele abolished reporter activity (p = .004) and reduced GATA binding (-14.5%). CRISPRa of the associated region increased RSPO4 expression (25.8 ± 6.5-fold, p = .003). RSPO4 activation showed strongest induction of Gliomedin (439-fold) and Mucin 21 (178-fold) and of the gene set "response to interferon-alpha" (area under the curve [AUC] = 0.8, p < 5 × 10-6 ). The most repressed gene set was "extracellular matrix interactions" (AUC = 0.8, padj = .00016). CONCLUSION: RSPO4 is a potential periodontitis risk gene and modifies host defence and barrier integrity.
Asunto(s)
Pérdida de Hueso Alveolar , Periodontitis , Animales , Ratones , Moléculas de Adhesión Celular Neuronal , Genotipo , Inmunidad Innata/genética , Periodontitis/genética , HumanosRESUMEN
AIM: The basis of phenotypic variation of periodontitis is genetic variability. Disease relevant effects of individual risk alleles are considered to result from genetic interactions. We investigated gene × gene (G×G) interactions of suggestive periodontitis susceptibility alleles. MATERIALS AND METHODS: We used the case-only design and investigated single-nucleotide polymorphism (SNPs) that showed associations in our recent genome-wide association study (GWAS) and GWAS meta-analysis with p < 5 × 10-6 . CRISPR-dCas9 gene activation followed by RNA-sequencing and gene-set enrichment analyses elucidated differentially expressed genes and gene networks. With the databases of SNPInspector and Transfac professional, luciferase reporter gene assays and antibody electrophoretic mobility shift experiments, we analysed allele-specific effects on transcription factor binding. RESULTS: SNPs at the genes sialic acid binding Ig-like lectin 5 (SIGLEC5) and plasminogen (PLG) showed G×G interactions with rs1122900 at the long non-coding RNA (lncRNA) CTD-2353F22. Associated chromatin cis-activated CTD-2353F22.1 6.5-fold (p = .003), indicating CTD-2353F22.1 as target gene of this interaction. CTD-2353F22.1 regulated GADD45A (padj < 4.9 × 10-11 , log2 fold change (FC) = -0.55), THBS1, SERPINE1 and Tissue Factor F3 (padj < 5 × 10-7 , log2 FC ≥ -0.35) and the gene set "angiogenesis" (area under the curve = 0.71, padj = 8.2 × 10-5 ). rs1122900 effect C-allele decreased reporter gene activity (5.5-fold, p = .0003) and PRDM14 binding (76%). CONCLUSIONS: CTD-2353F22.1 mediates interaction of SIGLEC5 and PLG, together with genes that function in periodontal wound healing.
Asunto(s)
Estudio de Asociación del Genoma Completo , ARN Largo no Codificante , Humanos , ARN Largo no Codificante/genética , Plasminógeno/genética , Polimorfismo de Nucleótido Simple/genética , Cicatrización de Heridas , Predisposición Genética a la Enfermedad/genética , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos CD/genética , Lectinas/genéticaRESUMEN
Endothelialization of blood contacting implants, e.g., vascular stents, is regarded as a prerequisite for an improved performance in terms of minimizing thrombogenicity and the inhibition of restenosis. Commonly used materials, such as Ti-based alloys, can be surface-modified in order to improve endothelial cell (EC) colonization as well as to reduce platelet adhesion. Standard modification techniques involve silanization and are laborious and time-consuming. We propose a novel single-step procedure based on a surface-recognizing peptide generated by phage display methodology. Combining this with a polyethylene glycol (PEG) spacer and an EC-specific sequence yielded a conjugate applicable for the modification of Ti surfaces.
Asunto(s)
Materiales Biocompatibles Revestidos/química , Endotelio Vascular/citología , Péptidos/química , Titanio/química , Plaquetas/citología , Adhesión Celular , Línea Celular , Materiales Biocompatibles Revestidos/efectos adversos , Humanos , Péptidos/efectos adversos , Polietilenglicoles/efectos adversos , Polietilenglicoles/química , Stents/efectos adversos , Propiedades de Superficie , Trombosis/etiología , Trombosis/prevención & control , Titanio/efectos adversosRESUMEN
The mating type (MAT) locus is the key regulator of sexual reproduction in fungi. In the dermatophytes and other Ascomycetes this genomic region exists in two distinct forms (idiomorphs) and their balanced presence is a precondition for successful mating in heterothallic fungi. But the MAT locus not only drives sexual reproduction, it has also been shown to influence pathogenicity, virulence, and/or morphological changes in pathogenic fungi of the genera Candida, Histoplasma, and Cryptococcus. In order to find out whether there are similar trends in dermatophytes, we investigated the MAT locus of 19 anthropophilic and zoophilic species via Sanger sequencing and primer walking. We identified for the first time the MAT locus idiomorphs of the dermatophyte species Microsporum audouinii (MAT1-2), M. ferrugineum (MAT1-2), Trichophyton schoenleinii (MAT1-2), T. bullosum (MAT1-1), T. quinckeanum (MAT1-1), T. concentricum (MAT1-1), T. eriotrephon (MAT1-1), and T. erinacei (MAT1-2). In addition, we determined the MAT locus sequence for dermatophyte species whose mating type idiomorphs had been described on the basis of results of classical confrontation experiments (e.g. M. canis, MAT1-2) and we confirmed recently published molecular data (e.g. T. rubrum, MAT1-2). Our results corroborate that MAT locus idiomorphs are unequally distributed in the majority of the analyzed species and the ability to mate with a partner of the opposite sex is limited to a few zoophilic species. Clonal spreads are identified that are connected to one of the idiomorphs and a higher virulence and/or a higher transmission rate to humans (T. benhamiae and T. mentagrophytes). For the imbalanced idiomorph distribution pattern we hypothesize that either: (I) one of the mating type idiomorphs may be extinct due to clonal reproduction (e.g., T. rubrum and M. canis), (II) mating partners of one species adapted to different hosts followed by speciation in the new niche (e.g., T. equinum and T. tonsurans) or (III) unisexual reproduction is the next evolutionary stage of propagation in dermatophytes which involves the extinction of one mating idiomorph.