Fig. 5

LncRNA H19 binds miR-21a-5p to regulate the expression of samd7. (A) BMP2, BMP2 + simH19 induced changes in miRNA expression. C3H10T1/2 cells were infected with AdBMP2 or AdBMP2+AdsimH19 and micro cultured. The changes of miRNA expression in C3H10T1/2 cells were detected by microarray at 12 days after infection, and the results showed significant changes in miRNA expression. (B) microarray results showed that eight miRNAs were significantly up-regulated in the AdBMP2+simH19 group, among which miR-21a-5p was increased threefold compared with the control group. (C) AdsimH19 increased the expression of miR-21a-5p in chondrocytes. qPCR was used to detect the expression of miR-21a-5p in cultured mouse chondrocytes infected with AdBMP2 or AdBMP2+AdsimH19 in vitro. Expression of miR-21a-5p at day 3, day 7 and day12 showed increased in the AdBMP2+AdsimH19 group. D, H19 wild type, miR-21a-5p simulator and H19 mutant double luciferase gene sequence. (E) H19 and miR-21a-5p have binding sites. Luciferase activity of HEK293 cells transfected with NC mimics + H19 wt, mmu-miR-21a-5p + H19 wt, NC mimics + H19 mu and mmu-miR-21a-5p + H19 mu was detected. The results showed that compared with the NC mimics + H19 wt group, the luciferase activity of the mmu-miR-21a-5p + H19 wt group was decreased, and compared with the mmu-miR-21a-5p + H19 wt group, the luciferase activity of the mmu-miR-21a-5p + H19 mu group was increased. “*” means p < 0.05, “**” means p < 0.01. (F) BMP2-induced mRNA expression of H19 and smad7. Stimulated by AdBMP2, the relative expression levels of H19 and smad7 were measured by reverse transcription and quantitative real-time PCR (RT-qPCR) at a series of times (days 1, 2, 3, 5, 7, 9, and 12). AdGFP was used as a control. Values for AdBMP2 / AdGFP are shown (each assay was triplicate and/or performed in at least three separate experiments).