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Objective To investigate the effect of microRNA (miR)-295 on high glucose-induced apoptosis of mouse osteoblast cell line(MC3T3-E1) and underlying the mechanism. Methods MC3T3-E1 cells cultured in vitro were divided into three groups: control (Ctrl) group, high glucose (HG) group (induced by 22 mmol/L glucose), (HG + miR-control) group (induced by high glucose after miR-control transfection) and (HG+miR-295) group (induced by high glucose after transfection of miR-295 mimic). The expression of miR-295 in MC3T3-E1 cells was detected by real-time fluorescent quantitative PCR, the survival rate of MC3T3-E1 cells was detected by methyl thiazolyl tetrazolium (MTT) assay, the apoptosis rate of MC3T3-E1 cells was detected by flow cytometry, the protein expression of Wnt/β-catenin pathway related proteins β-catenin, Dickkopf homolog 1 (DKK1), c-Myc and B-cell lymphoma-2 (Bcl-2) were detected by Western blot, double luciferase reporter gene assay was used to detect the targeting relationship between miR-295 and DKK1. Results Compared with those in the Ctrl group, the expression levels of miR-295, cell survival rate and the protein expression levels of β-catenin, c-Myc and Bcl-2 in MC3T3-E1 cells in HG group were significantly lower, the apoptosis rate and protein expression level of DKK1 were significantly higher (P＜0.05); Compared with those in (HG + miR-control) group, the expression level of miR-295 (P＜0.05), cell survival rate and the protein expression levels of β-catenin, c-Myc and Bcl-2 in MC3T3-E1 cells in (HG + miR-295) group were significantly higher (P＜0.05), the apoptosis rate and protein expression level of DKK1 were significantly lower(P＜0.05). Dkk1 might be the target gene of miR-295. Conclusions miR-295 may inhibit high glucose-induced MC3T3-E1 cell apoptosis by down-regulating DKK1 expression and activating Wnt/β-catenin pathway.