miR-642a-3p通过HIF-1α/EGFR通路参与破骨细胞分化的机制研究

The mechanistic study of miR-642a-3p promotes osteoclast differentiation through the hypoxia-inducible factor 1α/epidermal growth factor receptor pathway

摘要:

目的探讨miR-642a-3p对破骨细胞分化的影响及其机制。 方法采用THP-1细胞系，经PMA(100ng/mL)诱导24h分化为单核巨噬细胞后，联合RANKL(50ng/mL)和M-CSF(30ng/mL)分别诱导3、5、8d建立破骨细胞分化模型。通过慢病毒转染构建miR-642a-3p敲低(Sh-miR-642a-3p)细胞模型；采用骨吸收陷窝实验、TRAP染色评估破骨细胞骨吸收能力；qRT-PCR和Western印迹法检测miR-642a-3p、破骨细胞分化标志基因(NFATc1、CTSK、ACP5)、破骨细胞特异性蛋白(MMP9、NFATc1、CTSK)及通路关键分子(EGFR、HIF-1α)的表达；结合生物信息学(TargetScan、miRDB、DisGeNET数据库及GSE35958数据集)预测miR-642a-3p靶基因及调控通路。 结果破骨细胞分化过程中，骨吸收陷窝面积呈时间依赖性显著增加(第8天达峰值，P<0.01)。TRAP染色显示，第8天的破骨细胞较大，数量多且胞质颜色深、破骨细胞分化标志基因mRNA及破骨细胞特异性蛋白水平呈时间依赖性上调(第8天达峰值，P<0.001)，miR-642a-3p表达水平随诱导时间显著升高(第8天达峰值，P<0.05)。敲低miR-642a-3p后，骨吸收陷窝面积减少(P<0.01)，破骨细胞数量明显减少(P＜0.01)，破骨细胞分化标志基因及破骨细胞特异性蛋白表达均显著下调(P<0.05)。生物信息学分析显示，miR-642a-3p调控HIF-1α通路，其中EGFR是骨质疏松差异表达关键基因(P<0.05)。 qRT-PCR和Western印迹法结果显示，EGFR、HIF-1α在破骨细胞分化过程中表达上调，而敲低miR-642a-3p后两者表达同步抑制(P<0.05)。 结论miR-642a-3p通过激活HIF-1α/EGFR信号通路促进破骨细胞分化，可能成为绝经后骨质疏松症的潜在治疗靶点。

Abstract:

ObjectiveTo investigate the effect of miR-642a-3p on osteoclast differentiation and its underlying mechanism. MethodsThe THP-1 cell line was first induced to differentiate into macrophages using phorbol-12-myristate-13-acetate(PMA, 100ng/mL) for 24 hours. Subsequently, an osteoclast differentiation model was established by treating these macrophages with receptor activator of nuclear factor kappa-B ligand(RANKL, 50ng/mL) and macrophage colony-stimulating factor(M-CSF, 30ng/mL) for 3,5, and 8 days, respectively. The miR-642a-3p knockdown(Sh-miR-642a-3p) cell model was constructed via lentiviral transduction. Osteoclast bone resorption capacity was evaluated using the bone resorption pit assay and TRAP staining. Quantitative real-time PCR(qRT-PCR) and Western blotting(WB) methods were performed to detect the expression levels of miR-642a-3p, osteoclast differentiation marker genes(NFATc1, CTSK, ACP5), osteoclast-specific proteins(MMP9, NFATc1, CTSK), and key signaling molecules [epidermal growth factor receptor(EGFR), hypoxia-inducible factor(HIF)-1α]. Bioinformatics analysis(utilizing TargetScan, miRDB, DisGeNET databases, and the GSE35958 dataset) was employed to predict miR-642a-3p target genes and associated regulatory pathways. ResultsDuring osteoclast differentiation, the area of bone resorption pits increased significantly in a time-dependent manner(peaking on day 8, P<0.01). The TRAP staining showed that the osteoclasts were larger, more numerous, and dark in cytoplasm on day 8. Concurrently, the mRNA levels of osteoclast differentiation marker genes and the protein levels of osteoclast-specific markers were significantly up-regulated over time(peaking on day 8, P<0.001). The expression level of miR-642a-3p also significantly increased with induction time(peaking on day 8, P<0.05). Knockdown of miR-642a-3p resulted in a significant reduction in the resorption pit area(P<0.01), the number of osteoclasts was significantly reduced, and the expression of both osteoclast differentiation marker genes and osteoclast-specific proteins were down-regulated(P<0.05). The bioinformatics analysis indicated that miR-642a-3p regulated the HIF-1α pathway, with EGFR identified as a key differentially expressed gene(DEG) associated with osteoporosis(P<0.05). Experimental validation confirmed that the expression of both EGFR and HIF-1α were up-regulated during osteoclast differentiation, and this up-regulation was synchronously suppressed upon miR-642a-3p knockdown(P<0.05). ConclusionmiR-642a-3p promotes osteoclast differentiation by activating the HIF-1α/EGFR signaling pathway, and could be a potential therapeutic target for postmenopausal osteoporosis.