Department of Spine Surgery, The First Affiliated Hospital, Hengyang Medical School, University of South China, Hengyang, Hunan, China.
The First Clinical Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, China.
Cell Cycle. 2022 Feb;21(4):323-339. doi: 10.1080/15384101.2021.2020042. Epub 2022 Jan 2.
Intervertebral disc degeneration (IDD) constitutes the pathological foundation of most musculoskeletal disorders of the spine. Previous studies have noted that cell proliferation is a common feature of IDD. Bioinformatics indicated that aberrantly expressed long non-coding RNAs (lncRNAs) were involved in the development of IDD. In this study, we aimed to investigate the function of lncRNA HOTAIR in the proliferation of human nucleus pulposus (NP) cells of IDD in vitro and further clarified its mechanism. The expression of HOTAIR and miR-130b was quantified by qRT-PCR in nucleus pulposus (NP) tissues. Furthermore, NP cells proliferation were assayed by CCK8 and Immunostaining. Dual-luciferase reporter and RIP assay were used to examine the expression of HOTAIR, PTEN, and their co-target gene miR-130b. Western blotting was used to test AKT expression. Our experiments on human normal NP cells observed that HOTAIR was significantly dysregulated in IDD. Further, HOTAIR can suppress proliferation by directly targeting miR-130b. In addition, Both HOTAIR and PTEN were confirmed to target miR-130b, and miR-130b upregulation reversed the phenomenon of ectopic expression of HOTAIR. More importantly, HOTAIR upregulation significantly reduced CyclinD1 protein expression by PTEN/AKT signaling pathway. Our findings suggest that HOTAIR may bind to miR-130b and subsequently increased CyclinD1 expression via PTEN/Akt pathway. Thereby, HOTAIR could become a potential target for the treatment of IDD. IDD; intervertebral disc degeneration ncRNAs; non-coding RNAs lncRNAs; long non-coding RNAs miRNAs; microRNAs NP; nucleus pulposus qRT-PCR; quantitative reverse transcription-PCR LBP; Low back pain ORF; open reading frame HOTAIR; Hox transcript antisense intergenic RNA FAF1; Fas-associated protein factor-1 Erk; extracellular signal-regulated kinase TUG1; Taurine Up-regulated Gene 1 HIF1A hypoxia-inducible factor 1-alpha PI3K; phosphoinositide-3 kinase AIS; adolescent idiopathic scoliosis ECM; extracellular matrix LN;lupus nephritis CT;computed tomography MRI; magnetic resonance imaging PBS; phosphate-buffered salin PBS; phosphate-buffered salin PVDF; polyvinylidene fluoride TBST; Tris-buffered saline Tween ECL; enhanced chemiluminescence RIP; RNA immunoprecipitation.
椎间盘退变性（IDD）构成了脊柱大多数肌肉骨骼疾病的病理基础。先前的研究表明，细胞增殖是 IDD 的共同特征。生物信息学表明，异常表达的长非编码 RNA（lncRNA）参与了 IDD 的发展。在这项研究中，我们旨在研究 lncRNA HOTAIR 在体外 IDD 人髓核（NP）细胞增殖中的作用，并进一步阐明其机制。通过 qRT-PCR 定量检测核髓核（NP）组织中 HOTAIR 和 miR-130b 的表达。进一步通过 CCK8 和免疫染色法检测 NP 细胞增殖。双荧光素酶报告和 RIP 测定用于检测 HOTAIR、PTEN 及其共同靶基因 miR-130b 的表达。Western blot 用于检测 AKT 表达。我们对人正常 NP 细胞的实验观察到，IDD 中 HOTAIR 明显失调。此外，HOTAIR 可通过直接靶向 miR-130b 抑制增殖。此外，HOTAIR 和 PTEN 均被证实可靶向 miR-130b，miR-130b 的上调逆转了 HOTAIR 异位表达的现象。更重要的是，HOTAIR 上调通过 PTEN/AKT 信号通路显著降低了 CyclinD1 蛋白表达。我们的研究结果表明，HOTAIR 可能通过与 miR-130b 结合，进而通过 PTEN/Akt 通路增加 CyclinD1 的表达。因此，HOTAIR 可能成为治疗 IDD 的潜在靶点。IDD；椎间盘退变性 ncRNAs；非编码 RNA lncRNAs；长非编码 RNA miRNAs；microRNAs NP；核髓核 qRT-PCR；定量逆转录 PCR LBP；下腰痛 ORF；开放阅读框 HOTAIR；同源盒转录反义基因间 RNA FAF1；Fas 相关蛋白因子-1 Erk；细胞外信号调节激酶 TUG1；牛磺酸上调基因 1 HIF1A 缺氧诱导因子 1-α PI3K；磷酸肌醇-3 激酶 AIS；青少年特发性脊柱侧凸 ECM；细胞外基质 LN；狼疮肾炎 CT；计算机断层扫描 MRI；磁共振成像 PBS；磷酸盐缓冲盐水 PBS；磷酸盐缓冲盐水 PVDF；聚偏二氟乙烯 TBST；Tris 缓冲盐 Tween ECL；增强化学发光 RIP；RNA 免疫沉淀。
