lncRNA CDKN2B-AS1 regulates collagen expression

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=9.7x10-5 (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 x 10-25). The inflammatory "TNFA signaling via NFKB" gene set was downregulated the most (Padj=1x10-5 (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<9x10-24). The risk variant rs10757278 altered a binding site of the pathogen responsive transcription factor STAT1 (P=5.8x10-6). 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. To establish the molecular function of the lncRNA CDKN2B-AS1, we downregulated CDKN2B-AS1 transcript levels in cultures of primary gingival fibroblasts from 3 different donors with LNA GapmeRs. We then performed gene expression profiling by RNA-Sequencing with RNA extracted 48 hours after LNA GapmeR transfection. 3 control samples were transfected with negative control GapmeRs and matched with the 3 samples that were transfected with LNA GapmeRs that targeted CDKN2B-AS1. Additionally, we transfected an immortalized (hTERT) human gingival fibroblast cell line (ihGF; Applied Biological Materials Inc, cat.-no T0026) with LNA GapmeRs that targeted CDKN2B-AS1 and with negative control LNA GapmeRs ------------------------- Authors state "no file provided due to donor privacy concerns".

长链非编码RNA（long noncoding RNA，lncRNA）CDKN2B-AS1携带一个主要的冠心病风险单倍型，该单倍型同时与进展性口腔炎症性疾病牙周炎以及心肌梗死（myocardial infarction，MI）相关。尽管已有大量研究，但目前学界尚未就CDKN2B-AS1的功能达成广泛共识，无法解释该长链非编码RNA在上述疾病中的共通分子作用机制。本研究旨在探究CDKN2B-AS1在牙龈细胞中的作用，以更好地理解进展性牙周炎风险升高背后的分子机制。我们利用LNA GapmeRs下调原代牙龈成纤维细胞中CDKN2B-AS1的转录本水平。完成RNA测序（RNA-sequencing）后，我们开展了差异表达分析、基因集富集分析以及蛋白质免疫印迹（Western Blotting）实验。我们通过分析相关DNA序列变异对预测转录因子结合位点的影响，搜寻潜在的致病等位基因。我们在牙龈成纤维细胞与HeLa细胞中，利用荧光素酶报告基因实验及抗体电泳迁移率变动分析对潜在功能等位基因进行了功能验证。在所分析的所有基因集中，胶原蛋白生物合成通路的上调幅度最为显著（校正后P值Padj=9.7×10⁻⁵，AUC>0.65），其中冠心病与心肌梗死风险基因COL4A1在富集基因集中的上调程度最高（折叠变化=12.13，Padj=4.9×10⁻²⁵）。而炎症相关的“通过NFKB的TNFA信号通路”基因集下调最为显著（Padj=1×10⁻⁵，AUC=0.60）。在单个基因层面，参与细胞外基质组织的CAPNS2是上调幅度最高的蛋白编码基因（折叠变化=48.5，P<9×10⁻²⁴）。风险变异rs10757278改变了病原体响应性转录因子STAT1的结合位点（P=5.8×10⁻⁶）。rs10757278-G等位基因可使STAT1结合能力降低14.4%，而rs10757278-A等位基因可使牙龈成纤维细胞中的荧光素酶活性降低41.2%（P=0.0056），这与GTEx数据库的数据相符。CDKN2B-AS1可抑制牙龈成纤维细胞中的胶原基因表达。在炎症因子刺激下，等位基因特异性的CDKN2B-AS1表达异常导致胶原蛋白生物合成失调，可能会影响胶原合成，进而影响组织屏障功能与动脉粥样硬化斑块稳定性。为明确长链非编码RNA CDKN2B-AS1的分子功能，我们利用靶向CDKN2B-AS1的LNA GapmeRs，下调3名不同供体来源的原代牙龈成纤维细胞的CDKN2B-AS1转录本水平。我们在LNA GapmeRs转染48小时后提取RNA，通过RNA测序开展基因表达谱分析。其中3份对照样本转染了阴性对照GapmeRs，并与3份转染靶向CDKN2B-AS1的LNA GapmeRs的样本进行配对。此外，我们还利用靶向CDKN2B-AS1的LNA GapmeRs以及阴性对照LNA GapmeRs，转染了永生化人牙龈成纤维细胞系（ihGF；Applied Biological Materials Inc，货号T0026）。作者声明“因供体隐私问题，未提供相关文件”。