Roles of miRNA‐1 and miRNA‐133 in the proliferation and differentiation of myoblasts in duck skeletal muscle

Abstract MicroRNA (miRNA)‐1 and miRNA‐133 are derived from the same bicistronic pairs with roles in skeletal muscle development. Many investigations have focused on the role of miRNA‐1 and miRNA‐133 in the regulation of skeletal muscle development in mammals and fish. However, the mechanisms of miRNA‐1 and miRNA‐133 underlying the differences in skeletal muscle development between different breeds are not well known. Our study found that the weights of body and breast at 42 days of age were greater in Cherry Valley ducks than in Putian ducks and the areas of breast muscle fibers increased with age; the areas of muscle fibers of Cherry Valley ducks were always greater than those of Putian ducks. Besides, quantitative reverse‐transcriptase polymerase chain reaction analysis revealed that relatively high levels of miRNA‐1 and miRNA‐133 were detected in heart, breast, and leg muscles compared with the liver, spleen, lung, kidney, and the expression levels of miRNA‐1 and miRNA‐133 remained stable in the embryo stage, and in the growth period, the fluctuation in miRNA expression levels in Putian ducks was considerably higher than that in Cherry Valley ducks, especially from 7 to 28 days. However, in the late growth period, the expression of miRNA‐1 and miRNA‐133 of Cherry Valley duck was higher than that of Putian duck, which may indicate that miRNA‐1 and miRNA‐133 play a more important role during the growth period. To determine the function of miRNA‐1 and miRNA‐133 in skeletal muscle development, we found that the overexpression of miRNA‐1, but not miRNA‐133, promoted fusion of adjacent myoblasts. By contrast, a repressor of miRNA‐1 promoted, whereas a miRNA‐133 inhibitor inhibited, myoblast proliferation. Accordingly, the expression levels of myocyte enhancer factor 2D ( MEF2D ) and myogenic differentiation ( MYOD ) were significantly increased by an miRNA‐1 mimic and the miRNA‐133 inhibitor. In addition, we found that the expression levels of miRNA‐1 significantly affected the expression of histone deacetylase 4 ( HDAC4 ), and miRNA‐133 affected serum response factor ( SRF ) and transforming growth factor β receptor 1 ( TGFBR1 ) levels. However, dual‐luciferase reporter assays revealed that only miRNA‐1 directly inhibited pGL‐ HDAC4 luciferase reporter activity, whereas miRNA‐133 did not affect pGL‐ SRF or pGL‐ TGFBR1 fluorescence activity. Taken together, these results suggest that miRNA‐1 targets HDAC4 to promote the differentiation of duck myoblasts and miRNA‐133 may affect SRF and TGFBR1 expression to promote proliferation, which indicates that miRNA‐1 and miRNA‐133 play different important roles in skeletal muscle development.