miR-320a 在绝经后骨质疏松症中高表达，通过降低 MAP9 抑制 PI3K/AKT 信号通路在 MC3T3E1 细胞中起负调控作用。
MiR-320a was highly expressed in postmenopausal osteoporosis and acts as a negative regulator in MC3T3E1 cells by reducing MAP9 and inhibiting PI3K/AKT signaling pathway.
机构信息
Department of Osteoarticular Surgery, Jining NO.1 People's Hospital, China.
Department of Endocrinology, Jining NO.1 People's Hospital, China.
出版信息
Exp Mol Pathol. 2019 Oct;110:104282. doi: 10.1016/j.yexmp.2019.104282. Epub 2019 Jul 10.
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.
背景
绝经后骨质疏松症（PMO）作为绝经后妇女的常见疾病，主要是由于雌激素缺乏引起的。miR-320a 已被发现可减弱成骨细胞功能并在骨质疏松症发生前诱导氧化应激。然而，miR-320a 在 PMO 中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
基于 GEO 数据库分析骨质疏松症患者中 miR-320a 和微管相关蛋白 9（MAP9）的表达水平。通过噻唑蓝溴化（MTT）法测定细胞活力。通过流式细胞术和 Western blot 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的表达。通过生物软件预测 miR-320a 和 MAP9 之间的相互作用，并通过双荧光素酶报告基因检测和挽救实验进行验证。通过 Western blot 检测 MC3T3-E1 细胞中 PI3K、p-PI3K、AKT 和 p-AKT 的表达水平。
结果
我们观察到 miR-320a 在 PMO 患者中过表达，并表现出对 MC3T3-E1 细胞活性和分化的抑制作用，以及对 MC3T3-E1 细胞凋亡的促进作用。MAP9 被验证为 miR-320a 的靶基因，并受 miR-320a 的负调控。基于 GEO 数据库，发现 MAP9 在 PMO 患者中的表达水平较低。挽救实验表明，下调 MAP9 可减轻 miR-320a 抑制剂对 MC3T3-E1 细胞活性和分化的促进作用，以及对 MC3T3-E1 细胞凋亡的抑制作用。从机制上讲，miR-320a/MAP9 可能通过调节 PI3K/AKT 信号通路参与 MC3T3-E1 细胞活力、分化和凋亡。
结论
我们的研究结果表明，miR-320a 通过靶向 MAP9 并调节 PI3K/AKT 信号通路促进 MC3T3-E1 细胞凋亡，抑制 MC3T3-E1 细胞活力和分化，为 miR-320a/MAP9 作为治疗和预防 PMO 的有前途的靶点提供了理论依据。
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