Department of Orthopaedics, Liuzhou Traditional Chinese Medical Hospital, Guangxi University of Chinese Medicine, Liuzhou, Guangxi 545001, P.R. China.
The Second Department of Orthopaedic Surgery, Changzheng Hospital, Second Military Medical University, Shanghai 200003, P.R. China.
Mol Med Rep. 2020 Dec;22(6):5251-5261. doi: 10.3892/mmr.2020.11603. Epub 2020 Oct 14.
Intervertebral disc (IVD) degeneration is a complicated physiological change involving cellular senescence, inflammation and the degradation of the extracellular matrix. Long non‑coding RNAs (lncRNAs) have been identified as new players in IVD degeneration. The present study aimed to identify lncRNAs implicated in IVD degeneration via the regulation of cellular senescence. In the present study, nucleus pulposus (NP) cells isolated from moderately degenerated IVD tissues exhibited a senescent phenotype with increased senescence rates, detected by senescence‑associated β‑galactosidase (SA‑β‑gal) staining, and reduced growth and migratory abilities. Microarray and target prediction analyses identified 353 differentially expressed lncRNAs, and 251 cis‑ and 2,170 trans‑acting targets in degenerated NP cells. Bioinformatic analyses revealed that these predicted targets were enriched in the regulation of response to DNA damage stimulus, positive regulation of cell cycle processes and interferon‑β production. In addition, a network of the top 10 upregulated and top 10 downregulated lncRNA targets was constructed, and two trans‑acting targets, C‑C motif chemokine ligand 5 (CCL5) and polyribonucleotide nucleotidyltransferase 1 (PNPT1) involved in aging or senescence, and their corresponding lncRNAs, lnc‑ST8SIA5‑1:2 and lnc‑HRK‑2:1, were identified. Reverse transcription‑quantitative PCR validation demonstrated that the two targets and two candidate lncRNAs were significantly upregulated in degenerated NP cells. Overexpression of lnc‑HRK‑2:1, with validated higher expression levels, in normal NP cells induced a senescent phenotype, with enhanced rates of senescence detected by SA‑β‑gal staining in cells, decreased growth and migratory abilities and improved expression levels of CCL5 and PNPT1. Collectively, these results suggested that upregulation of lnc‑HRK‑2:1 prompted NP cell senescence in IVD degeneration, which may be associated with increased expression levels of CCL5 and PNPT1.
椎间盘（IVD）退变是一种涉及细胞衰老、炎症和细胞外基质降解的复杂生理变化。长链非编码 RNA（lncRNA）已被鉴定为 IVD 退变中的新调控因子。本研究旨在通过调控细胞衰老来鉴定参与 IVD 退变的 lncRNA。本研究中，从中度退变的 IVD 组织中分离的髓核（NP）细胞表现出衰老表型，衰老相关β-半乳糖苷酶（SA-β-半乳糖）染色检测到衰老率增加，细胞生长和迁移能力降低。微阵列和靶基因预测分析鉴定出退变 NP 细胞中存在 353 个差异表达的 lncRNA，251 个顺式和 2170 个反式作用靶基因。生物信息学分析显示，这些预测靶基因富集在对 DNA 损伤刺激的反应、细胞周期过程的正向调控和干扰素-β产生的调控中。此外，构建了 top10 上调和 top10 下调 lncRNA 靶基因的网络，鉴定出 2 个反式作用靶基因，即 C-C 基序趋化因子配体 5（CCL5）和多核苷酸核苷酸转移酶 1（PNPT1），它们涉及衰老或衰老，以及它们对应的 lncRNA，lnc-ST8SIA5-1：2 和 lnc-HRK-2：1。逆转录定量 PCR 验证表明，在退变 NP 细胞中，这两个靶基因和两个候选 lncRNA 均显著上调。在正常 NP 细胞中过表达具有验证更高表达水平的 lnc-HRK-2：1，诱导衰老表型，通过 SA-β-半乳糖染色检测到细胞衰老率增加，细胞生长和迁移能力降低，CCL5 和 PNPT1 的表达水平提高。综上所述，这些结果表明，lnc-HRK-2：1 的上调促使 IVD 退变中的 NP 细胞衰老，这可能与 CCL5 和 PNPT1 的表达水平增加有关。
