关键词: 成骨分化, 机械刺激, 微小RNA-199a, 胰岛素样生长因子1

Abstract: PURPOSE: To investigate the expression change of microRNA (miR) - 199a in MC3T3-E1 cells stimulated by mechanical stretch and its mechanism of osteogenic differentiation. METHODS: MC3T3-E1 cells cultured in vitro were loaded with 12% stretch for 0, 3, 6, 12 and 24 hours, alkaline phosphatase (ALP) activity was detected by ALP activity test kit, the expressions of osteocalcin (OCN), osteoblast specific transcription factor osterix (OSX), Runt related transcription factor 2 (Runx2) mRNA and miR-199a were detected by real-time fluorescence quantitative PCR. MC3T3-E1 cells were divided into control group, stretch group, stretch + miR-NC group and stretch + miR-199a group, and the expressions of miR-199a, OCN, OSX, Runx2 mRNA and protein and ALP activity were observed after 12% stretch and transfection of miR-199a. Alizarin red S (ARS) staining was used to observe calcium nodule formation ability. The target relationship between miR-199a and insulin like growth factor-1 (IGF1) was detected by double luciferase reporter gene assay; in addition, the effect of miR-199a mimic on IGF1 mRNA and protein expression was detected by real-time fluorescent quantitative PCR and Western blot. SPSS 24.0 software package was used to analyze the data. RESULTS: Compared with those at the time point of 0 h, ALP activity and expression level of OCN, OSX and Runx2 mRNA of MC3T3-E1 cells at 3, 6, 12 and 24 hours after mechanical stretch stimulation were significantly higher, while the expression level of miR-199a was significantly lower(P<0.05), and the change was most significant at 12 h. Compared with those in the control group, the expression level of miR-199a was significantly lower in the stretch group, while ALP activity, the expression level of OCN, OSX and Runx2 mRNA and protein, calcium nodule formation level were significantly high in the stretch group(P<0.05); there was no significant difference in the above indexes between the stretch group and stretch + miR-NC group(P>0.05). Compared with stretch + miR-NC group, the expression level of miR-199a in stretch + miR-199a group was significantly higher; while ALP activity, OCN, OSX, Runx2 mRNA and protein expression level, calcium nodule formation level were significantly lower(P<0.05). miR-199a could targetedly bind to IGF1, and the expression level of IGF1 mRNA and protein in MC3T3-E1 cells was significantly reduced by miR-199a mimic(P<0.05). CONCLUSIONS: MiR-199a can inhibit the osteogenic differentiation of MC3T3-E1 cells induced by mechanical stretch stimulation, and its mechanism may be related to the targeted regulation of IGF1 expression.

Key words: Osteogenic differentiation, Mechanical stimulation, MicroRNA-199a, Insulin like growth factor-1