Department of Spine Surgery, The First Affiliated Hospital of Dali University, Dali City, PR China.
Department of Traumatology, The Second Affiliated Hospital of Kunming Medical University, Kunming City, PR China.
Autoimmunity. 2021 Sep;54(6):313-325. doi: 10.1080/08916934.2021.1922890. Epub 2021 Jun 29.
Although long non-coding RNA LINC00963 has been reported to play a crucial regulatory role in osteoporosis (OP), its specific mechanism has not been well studied. Cell viability of human bone marrow mesenchymal stem cells (hBMSCs) transfected with short hairpin RNA targeting LINC00963 (sh-LINC00963) and negative control (sh-NC) was analysed by cell counting kit-8 (CCK-8) assay. Alkaline phosphatase (ALP) activity in hBMSCs transfected with sh-LINC00963 and sh-NC after induction by osteogenic medium (OM) on day 7 was detected. The protein expression levels of osteocalcin (OCN) and osteopontin (OPN) in hBMSCs transfected with sh-LINC00963 and sh-NC during OM induction on day 3 were detected by western blot. The relationship among LINC00963, miR-760, and E26 transformation specific-1 (ETS1) was determined by bioinformatics analysis, luciferase reporter assay, and RNA-binding protein immunoprecipitation (RIP) assay. A rat model with OP was established to confirm the role of LINC00963 . The expression level of LINC00963 was much lower in hBMSCs isolated from the discarded femoral head tissues of OP patients compared with that in health patients. Meanwhile, the expression level of LINC00963 was significantly increased and the expression level of miR-760 was decreased in hBMSCs during osteogenic induction. LINC00963 could bind to the 3'-untranslated region (3'-UTR) of miR-760 and negatively regulate the expression of miR-760, then promote the osteogenic differentiation in hBMSCs. ETS1 was identified as a target of miR-760. Moreover, overexpression of LINC00963 obviously reduced bone mineral density (BMD) of the left femur in OP rats and alleviated OP progression Our results demonstrated that LINC00963 positively regulated the expression of ETS1 by directly targeting miR-760, and then promoted osteogenic differentiation of hBMSCs , and also attenuated OP progression , suggesting that LINC00963 might be a potential therapeutic target for OP.
虽然长链非编码 RNA LINC00963 已被报道在骨质疏松症 (OP) 中发挥关键调节作用，但它的具体机制尚未得到很好的研究。通过细胞计数试剂盒-8 (CCK-8) 分析转染 LINC00963 短发夹 RNA (sh-LINC00963) 和阴性对照 (sh-NC) 的人骨髓间充质干细胞 (hBMSC) 的细胞活力。检测转染 sh-LINC00963 和 sh-NC 的 hBMSC 在成骨培养基 (OM) 诱导第 7 天的碱性磷酸酶 (ALP) 活性。通过蛋白质印迹法检测转染 sh-LINC00963 和 sh-NC 的 hBMSC 在 OM 诱导第 3 天的骨钙素 (OCN) 和骨桥蛋白 (OPN) 的蛋白表达水平。通过生物信息学分析、荧光素酶报告基因测定和 RNA 结合蛋白免疫沉淀 (RIP) 测定确定 LINC00963、miR-760 和 E26 转化特异性 1 (ETS1) 之间的关系。建立 OP 大鼠模型证实 LINC00963 的作用。与健康患者相比，来自 OP 患者废弃股骨头组织分离的 hBMSC 中 LINC00963 的表达水平低得多。同时，在成骨诱导过程中，hBMSC 中 LINC00963 的表达水平显著增加，miR-760 的表达水平降低。LINC00963 可以与 miR-760 的 3'-非翻译区 (3'-UTR) 结合并负调控 miR-760 的表达，从而促进 hBMSC 的成骨分化。ETS1 被鉴定为 miR-760 的靶标。此外，LINC00963 的过表达明显降低了 OP 大鼠左侧股骨的骨密度 (BMD)，并减轻了 OP 的进展。我们的研究结果表明，LINC00963 通过直接靶向 miR-760 正向调节 ETS1 的表达，从而促进 hBMSC 的成骨分化，并减轻 OP 的进展，提示 LINC00963 可能是 OP 的潜在治疗靶点。
