State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, No. 14, Section 3 of RenMinNanlu, Chengdu, 610041 Sichuan China.
Cell Mol Biol Lett. 2019 Mar 28;24:23. doi: 10.1186/s11658-019-0146-0. eCollection 2019.
To investigate the dysregulation of microRNAs (miRNAs) during the differentiation of osteoclasts and the precise roles of miR-125a-5p in the differentiation of osteoclasts.
The cell model of RAW 264.7 osteoclast precursor cell differentiation induced by RANKL plus M-CSF stimulation was established. During the early stage of osteoclast differentiation, miRNA expression profiles were detected using the biochip technique and analyzed by cluster analysis. TargetScan, miRTarBase and miRDB database analysis was applied to find the key target genes of miR-125a-5p. A dual luciferase experiment was conducted to identify the direct target of miR-125a-5p. MiR-125a-5p mimic transfection and anti-miR-125-5p treatment were conducted to verify the role of miR-125q-5p in osteoclast differentiation. The levels of triiodothyronine receptor auxiliary protein (TRAP), matrix metallopeptidase 2 (MMP-2), MMP-9 and cathepsin K were analyzed by qRT-PCR and western blot assay. The expression levels of MMP-2 and MMP-9 were determined using western blotting and immunofluorescence assay. The migration and invasion of RAW 264.7 cells were assessed by wound healing and Transwell invasion assays. The proliferation of RAW 264.7 osteoclast precursor cells was detected using MTT assay.
There were 44 microRNAs differently expressed during the differentiation of RAW 264.7 osteoclast precursor cells into osteoclasts, 35 of which were up-regulated and 9 were down-regulated. By luciferase reporter assay, it was confirmed that the TNF receptor superfamily member 1B gene (TNFRSF1B) was the target gene of miR-125a-5p. Up-regulation of miR-125a-5p inhibited TNFRSF1B protein expression and promoted osteoclast differentiation whereas down-regulation of miR-125a-5p caused completely opposite results.
In conclusion, overexpression of miR-125a-5p suppresses the expression of TNFRSF1B and promotes osteoclast differentiation. These results reveal the crucial role of miR-125a-5p in the differentiation of osteoclasts.
研究破骨细胞分化过程中 microRNAs (miRNAs) 的失调以及 miR-125a-5p 在破骨细胞分化中的精确作用。
建立 RANKL 联合 M-CSF 刺激诱导 RAW 264.7 破骨前体细胞分化的细胞模型。在破骨细胞分化的早期，采用生物芯片技术检测 miRNA 表达谱，并通过聚类分析进行分析。应用 TargetScan、miRTarBase 和 miRDB 数据库分析寻找 miR-125a-5p 的关键靶基因。通过双荧光素酶实验鉴定 miR-125a-5p 的直接靶基因。转染 miR-125a-5p 模拟物和抗 miR-125a-5p 处理验证 miR-125a-5p 在破骨细胞分化中的作用。通过 qRT-PCR 和 Western blot 分析检测三碘甲状腺原氨酸受体辅助蛋白 (TRAP)、基质金属蛋白酶 2 (MMP-2)、MMP-9 和组织蛋白酶 K 的水平。通过 Western blot 和免疫荧光测定法测定 MMP-2 和 MMP-9 的表达水平。通过划痕愈合和 Transwell 侵袭实验评估 RAW 264.7 细胞的迁移和侵袭。通过 MTT 测定法检测 RAW 264.7 破骨前体细胞的增殖。
在 RAW 264.7 破骨前体细胞分化为破骨细胞的过程中，有 44 个 microRNAs 表达不同，其中 35 个上调，9 个下调。通过荧光素酶报告基因实验证实 TNF 受体超家族成员 1B 基因 (TNFRSF1B) 是 miR-125a-5p 的靶基因。miR-125a-5p 的上调抑制了 TNFRSF1B 蛋白的表达并促进了破骨细胞的分化，而 miR-125a-5p 的下调则导致了完全相反的结果。
综上所述，miR-125a-5p 的过表达抑制了 TNFRSF1B 的表达并促进了破骨细胞的分化。这些结果揭示了 miR-125a-5p 在破骨细胞分化中的关键作用。
