Fan Jian-Bo, Zhang Yingzi, Liu Wei, Zhu Xin-Hui, Xu Da-Wei, Zhao Jian-Ning, Cui Zhi-Ming
Department of Orthopaedics, The Second Affiliated Hospital of Nantong University, Nantong, China.
Department of Orthopaedics, Jinling Hospital, Nanjing Medical University, Nanjing, China.
Cell Physiol Biochem. 2018;51(1):31-45. doi: 10.1159/000495159. Epub 2018 Nov 15.
BACKGROUND/AIMS: Dexamethasone (Dex) induces injuries to human osteoblasts. In this study, we tested the potential role of the long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (Lnc-MALAT1) in this process.
Two established human osteoblastic cell lines (OB-6 and hFOB1.19) and primary human osteoblasts were treated with Dex. Lnc-MALAT1 expression was analyzed by quantitative real-time polymerase chain reaction assay. Cell viability, apoptosis, and death were tested by the MTT assay, histone-DNA assay, and trypan blue staining assay, respectively. AMP-activated protein kinase (AMPK) signaling was evaluated by western blotting and AMPK activity assay.
Lnc-MALAT1 expression was downregulated by Dex treatment in the established osteoblastic cell lines (OB-6 and hFOB1.19) and primary human osteoblasts. The level of Lnc-MALAT1 was decreased in the necrotic femoral head tissues of Dex-administered patients. In osteoblastic cells and primary human osteoblasts, forced overexpression of Lnc-MALAT1 using a lentiviral vector (LV-MALAT1) inhibited Dex-induced cell viability reduction, cell death, and apoptosis. Conversely, transfection with Lnc-MALAT1 small interfering RNA aggravated Dex-induced cytotoxicity. Transfection with LV-MALAT1 downregulated Ppm1e (protein phosphatase, Mg2+/ Mn2+-dependent 1e) expression to activate AMPK signaling. Treatment of osteoblasts with AMPKα1 short hairpin RNA or dominant negative mutation (T172A) abolished LV-MALAT1-induced protection against Dex-induced cytotoxicity. Furthermore, LV-MALAT1 induced an increase in nicotinamide adenine dinucleotide phosphate activity and activation of Nrf2 signaling. Dex-induced reactive oxygen species production was significantly attenuated by LV-MALAT1 transfection in osteoblastic cells and primary osteoblasts.
Lnc-MALAT1 protects human osteoblasts from Dex-induced injuries, possibly via activation of Ppm1e-AMPK signaling.
背景/目的：地塞米松（Dex）可诱导人成骨细胞损伤。在本研究中，我们测试了长链非编码RNA转移相关肺腺癌转录本1（Lnc-MALAT1）在此过程中的潜在作用。
用Dex处理两种已建立的人成骨细胞系（OB-6和hFOB1.19）以及原代人成骨细胞。通过定量实时聚合酶链反应分析Lnc-MALAT1的表达。分别通过MTT法、组蛋白-DNA法和台盼蓝染色法检测细胞活力、凋亡和死亡情况。通过蛋白质印迹法和AMPK活性测定评估AMP激活的蛋白激酶（AMPK）信号传导。
在已建立的成骨细胞系（OB-6和hFOB1.19）以及原代人成骨细胞中，Dex处理下调了Lnc-MALAT1的表达。在接受Dex治疗的患者坏死股骨头组织中，Lnc-MALAT1水平降低。在成骨细胞和原代人成骨细胞中，使用慢病毒载体（LV-MALAT1）强制过表达Lnc-MALAT1可抑制Dex诱导的细胞活力降低、细胞死亡和凋亡。相反，用Lnc-MALAT1小干扰RNA转染会加重Dex诱导的细胞毒性。用LV-MALAT1转染可下调Ppm1e（蛋白磷酸酶，Mg2+/Mn2+依赖性1e）的表达以激活AMPK信号传导。用AMPKα1短发夹RNA或显性负性突变（T172A）处理成骨细胞可消除LV-MALAT1诱导的对Dex诱导的细胞毒性的保护作用。此外，LV-MALAT1诱导烟酰胺腺嘌呤二核苷酸磷酸活性增加和Nrf2信号传导激活。在成骨细胞和原代成骨细胞中，LV-MALAT1转染可显著减弱Dex诱导的活性氧产生。
Lnc-MALAT1可能通过激活Ppm1e-AMPK信号传导保护人成骨细胞免受Dex诱导的损伤。
