临床研究发现14-3-3η通过未明确的机制与骨质疏松症相关。我们的目的是研究14-3-3η在骨质疏松症中的作用及其与 miRNA 的潜在关联。分析基因表达综合 (GEO) 和人类蛋白质图谱1数据库以检查 OP 中14-3-3η的 mRNA 和蛋白质表达。基于DAVID进行基因富集分析以探索14-3-3η的潜在机制。 miRWalk 用于预测相关 miRNA。采用R软件和SPSS软件进行统计分析。通过慢病毒载体转染， 14-3-3η在 BMSC 中过表达并敲低表达。 BMSCs是通过缺氧诱导的。 qRT-PCR和Western-Blot验证了mRNA和蛋白的表达。划痕实验检测骨细胞的迁移。免疫共沉淀和荧光素酶测定研究了 14-3-3η 靶蛋白和 miRNA。过表达并敲低 miRNA 以验证14-3-3η与靶基因的关系。免疫组织化学染色图像证实，骨质疏松症患者的14-3-3η mRNA 表达水平较低。功能分析揭示了 MAPK 相关级联的富集。 14-3-3η与 MAPK 家族蛋白和 5 个关键 miRNA（包括mir-142-3p）相关。此外，BMSCs中14-3-3η敲低增加了Hif-α、VEGF、BMP-2、OPN、OST和Runx2的mRNA和蛋白表达水平，并增强了细胞的迁移能力。缺氧条件下，Hif-α和BMP-2蛋白表达水平上调，而14-3-3η和MAPK3蛋白表达水平下调。 免疫共沉淀实验显示 14-3-3η 与 MAPK3 的结合减少。 14-3-3η敲低产生与缺氧诱导相同的结果。添加caspase3抑制剂并敲低14-3-3η再次阻止MAPK3被caspase3裂解并抑制BMP-2表达。此外，在缺氧条件下， miR-142-3P表达上调，荧光素酶检测显示14-3-3η是其靶基因。 miR-142-3P过表达降低了 14-3-3η 和 MAPK3 的 mRNA 和蛋白水平，同时增加了 BMP-2 的表达。 miR-142-3P敲除逆转了这些结果。 BMSC 成骨被14-3-3η抑制，而miRNA-142-3p通过抑制14-3-3η促进成骨。
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Mir-142-3P regulates MAPK protein family by inhibiting 14-3-3η to enhance bone marrow mesenchymal stem cells osteogenesis
Clinical studies have found 14-3-3η to be associated with osteoporosis through undefined mechanisms. We aimed to investigate the role of 14-3-3η in osteoporosis and its potential associations with miRNAs. The Gene Expression Omnibus(GEO) and Human Protein Atlas 1 databases were analyzed to examine both the mRNA and protein expression of 14-3-3η in OP. Gene enrichment analyses were performed to explore the underlying mechanism of 14-3-3η based on DAVID. miRWalk was used to predict the associated miRNAs. The statistics were analysed by R software and SPSS software. 14-3-3η was overexpressed and knock down expressed in BMSCs by lentiviral vector transfecting. And BMSCs were induced by hypoxia. qRT-PCR and Western-Blot verified the expression of mRNA and protein. Scratch assay detected the migration of osteocytes. Co-immunoprecipitation and luciferase assay studied the 14-3-3η targeted protein and miRNA. overexpression and knock down of miRNA to verify the relationship of 14-3-3η and target genes. The 14-3-3η mRNA expression level was low in patients with osteoporosis, as corroborated by immunohistochemical staining images. Functional analyses revealed enrichment of the MAPK-associated cascade. 14-3-3η was correlated with MAPK family proteins and five key miRNAs, including mir-142-3p. In addition, 14-3-3η knockdown in BMSCs increased the mRNA and protein expression levels of Hif-α, VEGF, BMP-2, OPN, OST, and Runx2, and enhanced the cells migration ability. Under hypoxic conditions, Hif-α and BMP-2 protein expression levels were upregulated, whereas those of 14-3-3η and MAPK3 were downregulated. Co-immunoprecipitation experiments showed decreased binding of 14-3-3η to MAPK3. 14-3-3η knockdown produced the same results as hypoxia induction. Adding caspase3 inhibitor and knocking down 14-3-3η again prevented MAPK3 cleavage by caspase3 and inhibited BMP-2 expression. Moreover, under hypoxic conditions, miR-142-3P expression was upregulated and luciferase assays revealed 14-3-3η as its target gene. miR-142-3P overexpression decreased mRNA and protein levels of 14-3-3η and MAPK3, while increasing BMP-2 expression. miR-142-3P knockdown reversed these results. BMSC osteogenesis was suppressed by 14-3-3η, whereas miRNA-142-3p promoted it through the inhibition of 14-3-3η.
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