Abstract

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 in vivo. 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 in vivo. Our results demonstrated that LINC00963 positively regulated the expression of ETS1 by directly targeting miR-760, and then promoted osteogenic differentiation of hBMSCs in vitro, and also attenuated OP progression in vivo, suggesting that LINC00963 might be a potential therapeutic target for OP.

摘要

尽管有报道称长链非编码 RNA LINC00963 在骨质疏松症 (OP) 中发挥关键调节作用，但其具体机​​制尚未得到很好的研究。通过细胞计数试剂盒 8 (CCK-8) 测定法分析用靶向 LINC00963 (sh-LINC00963) 和阴性对照 (sh-NC) 的短发夹 RNA 转染的人骨髓间充质干细胞 (hBMSC) 的细胞活力。在第7天用成骨培养基（OM）诱导后，检测用sh-LINC00963和sh-NC转染的hBMSC中的碱性磷酸酶（ALP）活性。通过蛋白质印迹法检测第 3 天 OM 诱导期间转染 sh-LINC00963 和 sh-NC 的 hBMSCs 中骨钙素 (OCN) 和骨桥蛋白 (OPN) 的蛋白表达水平。通过生物信息学分析确定 LINC00963、miR-760 和 E26 转化特异性-1 (ETS1) 之间的关系，荧光素酶报告基因测定和 RNA 结合蛋白免疫沉淀 (RIP) 测定。建立OP大鼠模型确认LINC00963的作用在体内。与健康患者相比，从 OP 患者丢弃的股骨头组织中分离出的 hBMSCs 中 LINC00963 的表达水平要低得多。同时，在成骨诱导过程中，hBMSCs中LINC00963的表达水平显着升高，miR-760的表达水平降低。LINC00963 可以与 miR-760 的 3'-非翻译区 (3'-UTR) 结合并负调节 miR-760 的表达，进而促进 hBMSCs 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 大鼠左股骨的骨矿物质密度 (BMD)，并缓解了体内 OP 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，然后在体外促进 hBMSCs 的成骨分化，并在体内减弱 OP 进展，表明 LINC00963 可能是 OP 的潜在治疗靶点。