MiR-146b expression during zebrafish maturation. RT (real-time)-PCR analyses. MiR-146b expression after 3 (3d; sample n = 30), 7 (7d; sample n = 30), and 14 (14d; sample n = 30) days post fecundation (A). Expression levels of miR-146b at 2 (sample n = 30) and 6 months (sample n = 30) (B) and in young (sample n = 30), middle-aged (sample n = 30), and old zebrafish (sample n = 30) (C). Osteogenic (runxa, runxb, sp7), chondrogenic (sox9), and the receptor for RANK-Ligand gene expression increased during the maturation of larvae (D). Runx2a and sox9 gene expression was downregulated in 6 months compared to 2 months old zebrafish (E). Osteogenic or chondrogenic differentiation associated genes lowered in caudal fin during the aging (E). Rank gene expression, involved in osteoclast activation, was higher in the caudal fin of middle-aged and old zebrafish (F). Data are shown as mean ± standard deviation (SD); * p < 0.05; ** p < 0.01; *** p < 0.005. (A): Calcein staining of zebrafish at 14 days post fertilization. (B): Alizarin staining of zebrafish at 6 months of age. In particular, at 14dpf the miR-146b increased more than 8-fold compared to 3 dpf (A). Therefore, we evaluated the miR-146b expression in adult caudal fin of zebrafish, a model system for the evaluation of molecular mechanisms involved in regeneration [18]. In particular, miR-146b expression was evaluated in young (n = 30; 10–12 months), middle-aged (n = 30; 20–24 months), and old (n = 30; 30–36 months) zebrafish samples.
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