MiR-216b Influences The Differentiation, Fusion And Survival Of Osteoclasts By Directly Targeting To Inhibitor The Expression Of ABCG1

Objective: To investigate whether the miR-216 b can inhibitor the expression of ABCG1 protein by targeting ABCG1 3’UTR, and observe the effect and the related mechanisms on osteoclast differentiation, fusion, survival after over-expression or silence of ABCG1.Methods: Micro RNAs which may possible target to bind to ABCG1, MiR-216 b conservation between different species, target genes, ABCG1 3’UTR sequence, miR-216 b combined with ABCG1 3’UTR free energy, binding sites and other basic information were predicted and analysed by using Biological Information Analysis Network(micro RNA.org) and online software. The formation of osteoclasts, calculate the number of TRAP+ mature osteoclasts, diameter and number of the nuclear fusion were detected and calculated through the TRAP staining after treated with miR-216 b mimics or miR-216 b inhibitor for different concentrations(0, 10, 20, 40, 80 nm) and different time(0, 12, 24, 48 h). The expression ofABCG1 m RAN was detected by RT-PCR, Western blot was used to determine ABCG1 protein. MiR-216 b mimics, miR-216 b inhibitor or miR-216 b inhibitor + ABCG1 si RNA were transfected into cells for 48 h. The formation of osteoclasts, calculate the number of TRAP+ mature osteoclasts, diameter and number of the nuclear fusion were detected and calculated through the TRAP staining. The levels of ABCG1 m RAN and protein were examined by RT-PCR and western blot, the cholesterol efflux and intracellular cholesterol level were detected with liquid scintillation counter.Results: Biological Information Analysis Network and the online software analysis results showed that miR-216 b is highly conservative in many species, multiple target web site found that miR-216 b can target to combine with ABCG1 3’UTR and then regulate the expression of its target genes. MiR-216 b could inhibit the expression of ABCG1 protein and m RNA, and in a time- and concentration- dependent manner of ABCG1 protein expression, which further increased the number of osteoclasts, diameter, number of nuclear fusion, and then promoted osteoclast differentiation, fusion, and survival at last. MiR-216 b inhibitor could promote the expression of ABCG1 protein and m RNA, and in a time- and concentration- dependent manner of ABCG1 protein expression, which further decreased the number of osteoclasts, diameter, number of nuclear fusion, and then suppressed osteoclast differentiation, fusion, and survival at last. MiR-216 b inhibitor and ABCG1 si RNA were transfected into cells confirmed that miR-216 b reduced the cholesterol efflux through down-regulating ABCG1 protein expression in cells.Conclusions: MiR-216 b promoted osteoclast differentiation, fusion, and survival via reducing cholesterol efflux through down-regulating ABCG1 expression in osteoclasts.