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目的 研究miR-216b在破骨细胞分化中的功能和靶基因,探讨其对破骨细胞胆固醇外流的影响.方法 建立RANKL刺激诱导RAW 264.7破骨细胞前体细胞分化的细胞模型.进行抗酒石酸酸性磷酸酶(TRAP)染色测定以评估破骨细胞分化.通过生物信息学分析和双荧光素酶报告基因预测和分析miR-216b与其靶基因ABCG13\'非翻译区(3\'UTR)的结合以及自由能.转染miR-216b模拟物或抑制剂以验证miR-216b在破骨细胞分化中的作用.液体闪烁计数用于测量来自RAW264.7巨噬细胞衍生的破骨细胞[3H]标记的胆固醇流出.通过高效液相色谱(HPLC)检测RAW264.7巨噬细胞中的脂质积累.实时定量PCR (RT-qPCR)和蛋白质印迹分析用于评估破骨细胞中ABCG1的转录和转录后水平.结果 当细胞用miR-216b模拟物转染时,破骨细胞的数量、破骨细胞的平均直径和融合指数显著增加,如抗酒石酸酸性磷酸酶阳性染色和显微镜测定所揭示.MiR-216b抑制剂显示出完全相反的结果,这为我们的发现提供了额外的证据.生物信息学分析和双荧光素酶报告基因检测表明,miR-216b靶向ABCG1的3\'UTR.进一步研究表明,miR-216b抑制破骨细胞中ABCG1的mRNA和蛋白质水平.此外,我们还发现ABCGl siRNA对ABCG1的沉默增加了破骨细胞的数量、破骨细胞的平均直径和融合指数.MiR-216b通过抑制ABCG1表达减少破骨细胞的胆固醇流出.结论 总的来说,这些研究表明miR-216b下调ABCG1表达并抑制破骨细胞胆固醇流出,从而扰乱胆固醇稳态并促进破骨细胞生成.“,”Objective To investigated the function and the target gene of miR-216b in osteoclast differentiation and explored its effect on osteoclast cholesterol effiux.Methods The cell model of RAW264.7 osteoclast precursor cell differentiation induced by RANKL stimulation was established.Tartrate-resistant acid phosphatase (TRAP) staining assay was conducted to evaluate osteoclasts differentiation.MiR-216b target gene,ABCG 13\'untranslated region (3\'UTR) sequence and free energy were predicted and analyzed by bioinformatics analyses and dual-luciferase reporter assays.MiR-216b mimic or inhibitor transfection was performed to verify the role of miR-216b in osteoclast differentiation.Liquid scintillation counting was used to measure[3H]-labeled cholesterol effiux from RAW264.7 macrophage-derived osteoclasts.The lipid accumulation in RAW264.7 macrophages was detected by high performance liquid chromatography (HPLC).Real-time quantitative PCR (RT-qPCR) and Western blot assays were used to assess the transcriptional and post-transcriptional levels of ABCG 1 in osteoclasts.Results Our results showed that the number of osteoclasts,the average diameter of osteoclasts and the fusion index were significantly increased when cells were transfected with miR-216b mimic,as revealed by tartrate-resistant acid phosphatase-positive staining and microscopy assay.MiR-216b inhibitor showed the complete opposite outcome which brought additional evidence to our findings.Bioinformatics analysis and dual-luciferase reporter assays showed that miR-216b targets the 3\'UTR ofABCG1.Moreover,miR-216b suppressed both the mRNA and protein levels of ABCG1 in osteoclasts.Besides,we found that silencing of ABCG1 by ABCG1 siRNA increased the number of osteoclasts,the average diameter of osteoclasts and the fusion index.MiR-216b reduced cholesterol effiux from osteoclasts by inhibiting ABCG1 expression.Conclusion Collectively,these findings suggest that miR-216b downregulates ABCG1 expression and inhibits osteoclast cholesterol effiux,which disturbs cholesterol homeostasis and promotes osteoclastogenesis.