Long noncoding RNA NAV2-AS5 relieves chondrocyte inflammation by targeting miR-8082/TNIP2 in osteoarthritis.

CELL CYCLE 2023, VOL. 22, NO. 7, 796–807 https://doi.org/10.1080/15384101.2022.2154554 RESEARCH PAPER Long noncoding RNA NAV2-AS5 relieves chondrocyte inflammation by targeting miR-8082/TNIP2 in osteoarthritis Pu Wanga, Yuhao Wangb, and Baoan Maa a Department of Orthopedics, The Second Affiliated Hospital of The Air Force Medical University (Tangdu Hospital of Fourth Military Medical University), Xi’an, Shaanxi, P.R. China; bState Key Laboratory of Cancer Biology, Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi ‘an, Shaanxi, P.R.China ABSTRACT ARTICLE HISTORY Osteoarthritis (OA) is a common chronic and frequently occurring orthopedic disease in the middle-aged and elderly individuals. Numerous studies have shown that long noncoding RNAs (lncRNAs) play major roles in various diseases. However, the potential molecular mechanism of action of NAV2-AS5 in OA remains unclear. The present study was designed to explore the influence of NAV2-AS5 on the progression of chondrocyte inflammation and its underlying molecular mechanisms. To simulate the inflammatory environment in OA, the human chondro cyte cell line was treated with LPS. Cell proliferation, cell cycle progression, and apoptosis were assessed using Cell Counting Kit-8, 5-ethynyl-2’-deoxyuridine analysis, and flow cytometry. Proliferation- and cycle-related proteins and the release of inflammatory factors were examined by western blot analysis and enzyme-linked immunosorbent assay. The downstream targets of NAV2-AS5 were determined using bioinformatics and confirmed by a luciferase reporter assay. In our study, patients with OA showed downregulation of NAV2-AS5, upregulation of miR-8082, and downregulation of TNFAIP3 interacting protein 2 (TNIP2). Moreover, we found that both over expression of NAV2-AS5 and miR-8082 inhibitor promoted cell proliferation, inhibited apoptosis, and released inflammatory cytokines in LPS-treated chondrocytes. MiR-8082 was predicted to be a target of both NAV2-AS5 and TNIP2. In addition, rescue experiments showed that silencing of TNIP2 reversed the effects of the miR-8082 inhibitor on proliferation, cell cycle, apoptosis, and inflammatory factors in sh-NAV2-AS5-treated chondrocytes. In conclusion, these findings indicate that NAV2-AS5 relieves chondrocyte inflammation by targeting miR-8082/TNIP2 in OA, which provides a new theoretical basis for OA therapy. Received 23 July 2021 Revised 8 September 2022 Accepted 7 November 2022 Introduction Osteoarthritis (OA) is a degenerative joint disease characterized by gradual loss of articular cartilage and subchondral bone, together with varying degrees of synovial inflammation [1]. Due to the aging population and increasing obesity rates, the incidence of OA has gradually risen [2]. The World Health Organization has reported that 9.6% of men and 18.0% of women over 60 years of age have OA [3]. OA, which mainly manifests as joint pain, swelling, and stiffness, can affect sleep and lead to mental discomfort. Meanwhile, as one of the main causes of disability in adults, OA seriously disturbs the lives of patients and reduces their quality of life [4]. At present, reliev ing pain, protecting against and delaying disease KEYWORDS LncRNA NAV2-AS5; miR8082; TNIP2; chondrocyte; inflammation progression, and maintaining joint function have become the mainstay treatment modalities for patients with OA. However, these treatment meth ods are limited in clinical settings. Therefore, understanding OA pathogenesis is essential. However, the pathogenesis of OA is complex, and its specific pathological mechanisms remain unclear. Therefore, exploring effective ways to pre vent and treat OA based on its pathogenesis repre sents a direction for future research. Previous studies have shown that OA is a noninflammatory joint disease. However, various reports have shown that multiple immune cells, pro-inflammatory cytokines, complement pro teins, and other parts of the immune system play crucial roles in the occurrences of OA [5]. During CONTACT Baoan Ma Department of Orthopedics, The Second Affiliated Hospital of The Air Force Medical University (Tangdu Hospital of Fourth Military Medical University), No.1 Xinsi Road, Xiwang Village, Baqiao District, Xi’an, Shaanxi 710038, P.R. China Supplemental data for this article can be accessed online at https://doi.org/10.1080/15384101.2022.2154554 © 2022 Informa UK Limited, trading as Taylor & Francis Group CELL CYCLE the development of OA, inflammation involving joint tissues includes the production of cytokines, low-grade cell infiltration, and activation of joint cells such as synovial cells and articular cartilage cells [6]. Studies have shown that interleukin (IL)1 binding to its cognate receptor induces matrix metalloproteinases to degrade articular cartilage, reduce proteoglycan and collagen synthesis, and induce cartilage and synovial cells to produce inflammatory mediators, including IL-6 and IL-8, further leading to chondrocyte destruction [7]. In addition, IL-1, IL-6, and tumor necrosis factor (TNF)-α are all highly expressed in patients with OA. The levels of IL-1, IL-6, and TNF-α, which are positively correlated with the severity of inflamma tion, may be used as reference indicators to deter mine disease progression in patients with OA [8,9]. Many studies have shown that the occurrence and development of OA are inseparable from long noncoding RNAs (lncRNAs), which play vital roles in physiological and pathological processes [10]. For instance, lncRNA MALAT-1 reduces IL-1βinduced cell apoptosis and matrix metabolism dis order, and inhibits inflammation in chondrocytes by blocking activation of the JNK signaling path way [11]. Chen et al. integrated the analysis of critical mRNAs and lncRNAs in OA and found NAV2-AS5 significantly differentially expressed mRNAs between patients with OA and healthy controls [12]. NAV2-AS5 is a long chain, noncoding RNA composed of 3,336 nucleotides, but novel data on the molecular mechanisms and functional roles of lncRNA NAV2-AS5 in OA have not yet been reported. This study explored the mechanism of action of lncRNA NAV2-AS5 in OA. We found that lncRNA NAV2-AS5 regulates inflammatory responses in OA through the miR8082/TNIP2 signaling pathway. Materials and methods Clinical samples and ethics statement Thirty male osteoarthritis patients with an average age of 53.45 ± 5.18 years old and thirty healthy males with an average age of 54.73 ± 6.15 years old were recruited from the Second Affiliated Hospital of the Air Force Medical University 797 during total knee replacement surgeries. This study was approved by the Ethics Committee of the Second Affiliated Hospital of the Air Force Medical University and carried out in accordance with the Declaration of Helsinki. All participants provided signed informed consent prior to the study. Cartilage tissue samples were collected and stored in −80°C freezer until usage. Cell culture and transfecti (...truncated)