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OTUD6B-AS1 Might Be a Novel Regulator of Apoptosis in Systemic Sclerosis.

ABSTRACT: 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-? (TGF?), 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 TGF?, 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.

SUBMITTER: Takata M

PROVIDER: S-EPMC6533854 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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OTUD6B-AS1 Might Be a Novel Regulator of Apoptosis in Systemic Sclerosis.

Takata Miki M Pachera Elena E Frank-Bertoncelj Mojca M Kozlova Anastasiia A Jüngel Astrid A Whitfield Michael L ML Assassi Shervin S Calcagni Maurizio M de Vries-Bouwstra Jeska J Huizinga Tom W TW Kurreeman Fina F Kania Gabriela G Distler Oliver O

Frontiers in immunology 20190517

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 biop ...[more]

PMID: 31156645

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Project description:BackgroundThe long noncoding RNA (lncRNA) OTUD6B antisense RNA 1 (OTUD6B-AS1) is oriented in an antisense direction to the protein-coding gene OTUD6B on the opposite DNA strand. TCGA database data show that the expression of the lncRNA OTUD6B-AS1 is downregulated and that OTUD6B-AS1 acts as an antioncogene in a variety of tumors. However, the expression and biological functions of the lncRNA OTUD6B-AS1 are still unknown in tumors, including clear cell renal cell carcinoma (ccRCC).MethodsThe expression level of OTUD6B-AS1 was measured in 75 paired human ccRCC tissue and corresponding adjacent normal renal tissue samples. The correlations between the OTUD6B-AS1 expression level and clinicopathological features were evaluated using the chi-square test. The effects of OTUD6B-AS1 on ccRCC cells were determined via MTT assay, clone formation assay, transwell assay, and flow cytometry. Furthermore, the impact of OTUD6B-AS1 overexpression on the activation of the Wnt/β-catenin signaling pathway was investigated. Finally, ACHN cells with OTUD6B-AS1 overexpression were subcutaneously injected into nude mice to evaluate the influence of OTUD6B-AS1 on tumor growth in vivo.ResultsIn this study, we found that the expression of the lncRNA OTUD6B-AS1 was downregulated in ccRCC tissue samples and that patients with low OTUD6B-AS1 expression had shorter overall survival than patients with high OTUD6B-AS1 expression, which showed that the different expression level of OTUD6B-AS1 indirectly correlated with survival of patients. Lentivirus-mediated OTUD6B-AS1 overexpression significantly decreased the proliferation of ccRCC cells and promoted the apoptosis of the cells. Furthermore, OTUD6B-AS1 overexpression partly inhibited cell migration and invasion. The overexpression of OTUD6B-AS1 decreased the activity of the Wnt/β-catenin pathway and suppressed the expression of epithelial-to-mesenchymal transition (EMT)-related proteins (E-cadherin, N-cadherin and Snail) in ccRCC cells. In addition, compared with the parental ACHN cells, OTUD6B-AS1-overexpressing ACHN cells injected into nude mice exhibited decreased tumor growth in vivo.ConclusionsTaken together, our findings present a road map for targeting the newly identified lncRNA OTUD6B-AS1 to suppress ccRCC progression in cell lines, and these results elucidate a novel potential therapeutic target for ccRCC treatment.

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OTUD6B-AS1 Might Be a Novel Regulator of Apoptosis in Systemic Sclerosis.

Publications

OTUD6B-AS1 Might Be a Novel Regulator of Apoptosis in Systemic Sclerosis.

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