BACKGROUND Postmenopausal osteoporosis (PMO), as a frequent disease in postmenopausal women, is mainly caused by the lack of estrogen. MiR-320a has been found to abate osteoblast function and induce oxidative stress before osteoporosis. However, studies on the downstream target gene and related signaling pathway of miR-320a in PMO are still obscure. This study aims to deal with these problems. METHODS The expression levels of miR-320a and microtubule-associated protein 9 (MAP9) in patients with osteoporosis were analyzed on the basis of the GEO database. The cells viability was determined by methylthiazolyl tetrazolium bromide (MTT). Flow cytometry and western blot were used to detect the cells apoptosis and the expression of apoptosis-related proteins, respectively. The cells differentiation-related proteins were detected by qRT-PCR and western blot. The interaction between miR-320a and MAP9 was predicted by biological software and verified by dual luciferase reporter assay and rescue assay. The expression levels of PI3K, p-PI3K, AKT and p-AKT in MC3T3-E1 cells were assessed by western blot. RESULTS We observed that miR-320a was over-expressed in PMO patients and exhibited inhibitory effects on MC3T3-E1 cells activity and differentiation, as well as promoting effects on MC3T3-E1 cells apoptosis. MAP9 was verified as a target gene of miR-320a and was negatively regulated by miR-320a. Based on the GEO database, MAP9 was found to be lower expressed in PMO patients. Rescue assay demonstrated that down-regulation of MAP9 could alleviate the promoting effects of miR-320a inhibitor on MC3T3-E1 cells activity and differentiation and the inhibitory effects of miR-320a inhibitor on MC3T3-E1 cells apoptosis. Mechanically, miR-320a/MAP9 possibly took part in MC3T3-E1 cells viability, differentiation and apoptosis via mediating PI3K/AKT signaling pathway. CONCLUSIONS Our outcomes demonstrated that miR-320a promoted MC3T3-E1 cells apoptosis, suppressed MC3T3-E1 cells viability and differentiation through targeting MAP9 and modulating PI3K/AKT signaling pathway, which provided theoretical support for miR-320a/MAP9 as promising targets for the treatment and prevention of PMO.

中文翻译：

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MiR-320a在绝经后骨质疏松症中高表达，并通过减少MAP9和抑制PI3K / AKT信号通路而在MC3T3E1细胞中充当负调节剂。

背景技术绝经后骨质疏松症（PMO）作为绝经后妇女中的常见疾病，主要是由于缺乏雌激素引起的。已发现，MiR-320a可以减轻成骨细胞的功能并在骨质疏松症之前诱导氧化应激。然而，关于PMO中miR-320a的下游靶基因和相关信号通路的研究仍不清楚。本研究旨在解决这些问题。方法根据GEO数据库分析骨质疏松患者miR-320a和微管相关蛋白9（MAP9）的表达水平。通过甲基噻唑基溴化四唑（MTT）测定细胞活力。流式细胞仪和蛋白质印迹法分别检测细胞凋亡和凋亡相关蛋白的表达。通过qRT-PCR和western blot检测细胞分化相关蛋白。miR-320a和MAP9之间的相互作用通过生物学软件进行了预测，并通过双重荧光素酶报告基因分析和拯救分析进行了验证。用western blot检测PI3K，p-PI3K，AKT和p-AKT在MC3T3-E1细胞中的表达水平。结果我们观察到miR-320a在PMO患者中过表达，并且对MC3T3-E1细胞的活性和分化具有抑制作用，并且对MC3T3-E1细胞凋亡具有促进作用。MAP9被证实是miR-320a的靶基因，并受miR-320a负调控。根据GEO数据库，发现MAP9在PMO患者中表达较低。救援实验表明，MAP9的下调可以减轻miR-320a抑制剂对MC3T3-E1细胞活性和分化的促进作用，以及miR-320a抑制剂对MC3T3-E1细胞凋亡的抑制作用。在机械上，miR-320a / MAP9可能通过介导PI3K / AKT信号通路参与了MC3T3-E1细胞的活力，分化和凋亡。结论我们的结果表明，miR-320a通过靶向MAP9和调节PI3K / AKT信号通路来促进MC3T3-E1细胞凋亡，抑制MC3T3-E1细胞存活和分化，这为miR-320a / MAP9作为有希望的治疗靶点提供了理论支持。和预防PMO。miR-320a / MAP9可能通过介导PI3K / AKT信号通路参与了MC3T3-E1细胞的活力，分化和凋亡。结论我们的结果表明，miR-320a通过靶向MAP9和调节PI3K / AKT信号通路来促进MC3T3-E1细胞凋亡，抑制MC3T3-E1细胞存活和分化，这为miR-320a / MAP9作为有希望的治疗靶点提供了理论支持。和预防PMO。miR-320a / MAP9可能通过介导PI3K / AKT信号通路参与了MC3T3-E1细胞的活力，分化和凋亡。结论我们的结果表明，miR-320a通过靶向MAP9和调节PI3K / AKT信号通路来促进MC3T3-E1细胞凋亡，抑制MC3T3-E1细胞存活和分化，这为miR-320a / MAP9作为有希望的治疗靶点提供了理论支持。和预防PMO。