Division of Orthopedics and Traumatology, Department of Orthopedics, Nanfang Hospital, Southern Medical University, Guangzhou, China.
Department of Spine Orthopedics, Liuzhou Traditional Chinese Medical Hospital, Liuzhou, China.
J Cell Mol Med. 2021 May;25(10):4572-4582. doi: 10.1111/jcmm.16216. Epub 2021 Mar 17.
In this study, we aimed to explore the molecular mechanisms underlying the development of osteoporosis in post-menopausal females. Real-time PCR was conducted to measure the expression of potential lncRNAs involved in the osteoporosis of post-menopausal females. In addition, Western blot and IHC assays were used to study the possible correlation among HOTAIR, miR-138 and TIMP1, while a computational analysis was carried out to predict the 'seed sequence' responsible for the binding between miR-138 and HOTAIR/TIMP1. Furthermore, luciferase reporter assays were conducted to validate the negative regulatory relationship between miR-138 and TIMP1/HOTAIR. To evaluate the effect of oestrogen on the function of HOATIR and its downstream effectors, luciferase activity was measured in cells cotransfected with different vectors or treated with different doses of oestrogen. The results of the luciferase assay were further validated by real-time PCR, Western blot, MTT assay and flow cytometry. Among the candidate lncRNAs, HOTAIR was the only lncRNA down-regulated in post-menopausal females. HOTAIR bound to miR-138 and negatively regulated its expression. Meanwhile, miR-138 could also bind to TIMP1 mRNA and reduce its expression. Furthermore, a dose-dependent up-regulation of HOTAIR was observed in cells treated with oestrogen, and the elevated HOTAIR increased the level of TIMP1 by targeting miR-138. In addition, oestrogen promoted cell viability and suppressed cell apoptosis, and effects of oestrogen were blocked by the silencing of HOTAIR. Therefore, it can be concluded that oestrogen deficiency could induce the apoptosis of osteoblasts and lead to osteoporosis in post-menopausal females via modulation of the HOTAIR/miR-138/TIMP1 signalling axis.
在这项研究中，我们旨在探讨绝经后女性骨质疏松症发展的分子机制。通过实时 PCR 测量参与绝经后女性骨质疏松症的潜在 lncRNA 的表达。此外，通过 Western blot 和 IHC 检测研究 HOTAIR、miR-138 和 TIMP1 之间的可能相关性，同时进行计算分析以预测负责 miR-138 与 HOTAIR/TIMP1 结合的“种子序列”。此外，通过荧光素酶报告基因检测验证 miR-138 与 TIMP1/HOTAIR 之间的负调控关系。为了评估雌激素对 HOATIR 及其下游效应物功能的影响，在共转染不同载体或用不同剂量雌激素处理的细胞中测量荧光素酶活性。荧光素酶测定的结果通过实时 PCR、Western blot、MTT 测定和流式细胞术进一步验证。在候选 lncRNA 中，HOTAIR 是绝经后女性下调的唯一 lncRNA。HOTAIR 与 miR-138 结合并负调控其表达。同时，miR-138 也可以与 TIMP1 mRNA 结合并降低其表达。此外，在用雌激素处理的细胞中观察到 HOTAIR 的剂量依赖性上调，并且通过靶向 miR-138 升高的 HOTAIR 增加了 TIMP1 的水平。此外，雌激素促进细胞活力并抑制细胞凋亡，并且 HOTAIR 的沉默阻断了雌激素的作用。因此，可以得出结论，雌激素缺乏可通过调节 HOTAIR/miR-138/TIMP1 信号通路诱导成骨细胞凋亡并导致绝经后女性骨质疏松症。
