Department of Rheumatology, Center of Experimental Rheumatology, University Hospital Zürich, Zurich, Switzerland.
Department of Molecular and Systems Biology, Geisel School of Medicine at Dartmouth, Hanover, NH, United States.
Front Immunol. 2019 May 17;10:1100. doi: 10.3389/fimmu.2019.01100. eCollection 2019.
Antisense long non-coding RNAs (AS lncRNAs) have increasingly been recognized as important regulators of gene expression and they have been found to play key roles in several diseases. However, very little is known about the role of AS lncRNAs in fibrotic diseases such as systemic sclerosis (SSc). Our recent screening experiments by RNA sequencing showed that ovarian tumor domain containing 6B antisense RNA1 (OTUD6B-AS1) and its sense gene OTUD6B were significantly downregulated in SSc skin biopsies. Therefore, we aimed to identify key regulators of OTUD6B-AS1 and to analyze the functional relevance of OTUD6B-AS1 in SSc. OTUD6B-AS1 and OTUD6B expression in SSc and healthy control (HC) dermal fibroblasts (Fb) after stimulation with transforming growth factor-β (TGFβ), Interleukin (IL)-4, IL-13, and platelet-derived growth factor (PDGF) was analyzed by qPCR. To identify the functional role of OTUD6B-AS1, dermal Fb or human pulmonary artery smooth muscle cells (HPASMC) were transfected with a locked nucleic acid antisense oligonucleotide (ASO) targeting OTUD6B-AS1. Proliferation was measured by BrdU and real-time proliferation assay. Apoptosis was measured by Caspase 3/7 assay and Western blot for cleaved caspase 3. While no difference was recorded at the basal level between HC and SSc dermal Fb, the expression of OTUD6B-AS1 and OTUD6B was significantly downregulated in both SSc and HC dermal Fb after PDGF stimulation in a time-dependent manner. Only mild and inconsistent effects were observed with TGFβ, IL-4, and IL-13. OTUD6B-AS1 knockdown in Fb and HPASMC did not affect extracellular matrix or pro-fibrotic/proinflammatory cytokine production. However, OTUD6B-AS1 knockdown significantly increased Cyclin D1 expression at the mRNA and protein level. Moreover, silencing of OTUD6B-AS1 significantly reduced proliferation and suppressed apoptosis in both dermal Fb and HPASMC. OTUD6B-AS1 knockdown did not affect OTUD6B expression at the mRNA level and protein level. Our data suggest that OTUD6B-AS1 regulates proliferation and apoptosis via cyclin D1 expression in a sense gene independent manner. This is the first report investigating the function of OTUD6B-AS1. Our data shed light on a novel apoptosis resistance mechanism in Fb and vascular smooth muscle cells that might be relevant for pathogenesis of SSc.
反义长非编码 RNA（AS lncRNA）已被越来越多地认为是基因表达的重要调控因子，它们在多种疾病中发挥关键作用。然而，关于 AS lncRNA 在纤维化疾病（如系统性硬化症（SSc））中的作用知之甚少。我们最近通过 RNA 测序的筛选实验表明，卵巢肿瘤结构域包含 6B 反义 RNA1（OTUD6B-AS1）及其有意义的基因 OTUD6B 在 SSc 皮肤活检中显著下调。因此，我们旨在确定 OTUD6B-AS1 的关键调节因子，并分析 OTUD6B-AS1 在 SSc 中的功能相关性。通过 qPCR 分析 SSc 和健康对照（HC）真皮成纤维细胞（Fb）在转化生长因子-β（TGFβ）、白细胞介素（IL）-4、IL-13 和血小板衍生生长因子（PDGF）刺激后 OTUD6B-AS1 和 OTUD6B 的表达。为了确定 OTUD6B-AS1 的功能作用，用靶向 OTUD6B-AS1 的锁核酸反义寡核苷酸（ASO）转染真皮 Fb 或人肺动脉平滑肌细胞（HPASMC）。通过 BrdU 和实时增殖测定测量增殖。通过 Caspase 3/7 测定法和裂解 caspase 3 的 Western blot 测量细胞凋亡。虽然在基础水平上 HC 和 SSc 真皮 Fb 之间没有记录到差异，但在 PDGF 刺激后，OTUD6B-AS1 和 OTUD6B 的表达在 SSc 和 HC 真皮 Fb 中均呈时间依赖性显著下调。TGFβ、IL-4 和 IL-13 仅观察到轻微且不一致的影响。OTUD6B-AS1 在 Fb 和 HPASMC 中的敲低不影响细胞外基质或促纤维化/促炎细胞因子的产生。然而，OTUD6B-AS1 的敲低显著增加了 Cyclin D1 的 mRNA 和蛋白水平的表达。此外，OTUD6B-AS1 的沉默显著降低了真皮 Fb 和 HPASMC 的增殖并抑制了细胞凋亡。OTUD6B-AS1 的敲低不影响 OTUD6B 在 mRNA 水平和蛋白水平的表达。我们的数据表明，OTUD6B-AS1 通过 Cyclin D1 的表达以独立于有意义基因的方式调节增殖和凋亡。这是第一个研究 OTUD6B-AS1 功能的报告。我们的数据为 Fb 和血管平滑肌细胞中的新型凋亡抵抗机制提供了线索，这可能与 SSc 的发病机制有关。
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