Identification of miR-210 as a new myo-miR in trout

In fish, few miRNAs have been shown to muscle specific and data on miRNA involved in myogenesis are scarce. In order to identify new miRNA involved in satellite cells differentiation, we used a methionin depletion/replenishment protocol to synchronize myogenic cell differentiation. Our results clearly validated that methionine removal (72H) from the culture medium strongly decreased myoD1 and myogenin expression indicating a differentiation arrest. In contrast, methionine replenishment rescued the expression of myoD1 and myogenin showing a resumption of the differentiation. Then, we performed a miRNA array analysis of myogenic cells from three conditions: in presence of methionine (CTRL), absence of methionine during 72h (Meth-) and absence of methionine during 48H following 24H of methionine replenishment (Meth -/+). A clustering analysis identified three clusters : the cluster I corresponds to miRNA upregulated only in Meth -/+ conditions; the cluster II corresponds to miRNA downregulated only in Meth -/+ conditions; the cluster III corresponds to miRNAs with high expression in control, low expression in absence of methionine (Meth -) and middle expression after methionine replenishment (Meth -/+). The cluster III was very interesting because it followed (?) the resumption and differentiation and contained 7 miRNAs with muscle-related function (e.g. miR-133a) and one (miR-210) with unknown function. Based on our already published miRNAs repertoire (Juanchich et al 2016), we confirmed that miR-133a had an expression only in white muscle and we showed that miR-210 had the highest expression in white muscle. We also showed that miR-210 expression was upregulated during differentiation of satellite cells suggesting that miR-210 was potentially involved in the differentiation of satellite cells in trout. Trout cells in methionine depleted DMEM, in methionine supplemented DMEM, and supplemented DMEM during 24h after a 48h methionine restriction period