| microRNAs(miRNAs)are short single-stranded noncoding RNAs with a length of about 22 bp and highly conserved during evolution.miRNAs are widely expressed in single cells and multicellular organisms,and regulate gene expression at the post transcriptional level.miRNA-1 and miRNA-133 are derived from the same bicistronic pairs,which play an important role in the regulation of skeletal muscle development.Whereas,the research of duck miRNA-1 and miRNA-133 on muscle development has not been reported.In this study,Cherry Valley ducks and Putian ducks(white feathers)with different body sizes were used as experimental subjects,and the tissue expression profile and expression patterns during growth and development of duck miRNA-1 and miRNA-133 were detected by the real-time qPCR.To study the functions of miRNA-1 and miRNA-133,we transfected miRNA mimics or inhibitors into duck myoblasts to understand its important role on skeletal muscle development in ducks.The main findings are as follows:Firstly,to explore the pattern of growth and development of muscle and muscle fiber of Cherry Valley ducks and Putian ducks,this study measured the phenotypic data such as breast muscle weight,leg muscle weight and the area of muscle fiber on 38D?42 D?5 D?49 D?56 D.The results showed that the 42-day-old Cherry Valley ducks breast muscle weighted 205 g,leg muscle weighted 238 g,the area of muscle fiber of breast muscle and leg muscle were 5835 ?m2 and 12406 ?m2 respectively.While 42-day-old Putian ducks breast muscle weighted129 g,leg muscle weighted 175 g,and the area of muscle flber were 926 ?m2 and 4089 ?m2 respectively.The phenotypic data indicated that there was significant difference on the development of breast muscle,leg muscle and muscle fiber between Cherry Valley ducks and Putian ducks.Secondly,to construct the tissue expression profile and expression patterns during growth and development of duck miRNA-1 and miRNA-133,we detected the expression of miRNA-1 and miRNA-133 from Cherry Valley ducks and Putian ducks by real-time qPCR.The expression of miRNA-1 and miRNA-133 in breast and leg muscles showed a similar trend in the embryonic phase and postnatal stage of growth and development of Cherry Valley ducks and Putian ducks,which was,the miRNA expression increased sharply in the late embryonic stage,and was stable in the whole growth period after hatching.The expression level of Cherry Valley ducks respectively reached the peaks at the 28th day of embryos(E 28)and 42 days of growth period(42 D)and was significantly higher than that of Putian duck(P<0.05).Thirdly,to investigate the effect of miRNA-1 and miRNA-133 on development of skeletal muscle,miRNA-1 mimic or inhibitor and miRNA-133 mimic or inhibitor were transfected into duck myoblasts.Real-time qPCR analysis showed that miRNA mimic or inhibitor could effectively promote or depress its expression.Overexpression of miRNA-1 could promote the fusion of adjacent myoblasts,while fusion phenomenon was rare under the overexpression of miRNA-133,Cell counting test showed that inhibiting the miRNA-1 expression could promote myoblast proliferation,and inhibiting the miRNA-133 expression could inhibit the proliferation of myoblasts.The results of expression of myoblast differentiation marker genes,MEF2d and Myod,showed that,the expression of MEF2d and Myod were significantly increased after the transfection of miRNA-1 mimic and miRNA-133 inhibitor.These results suggested that miRNA-1 could promote the differentiation of duck myoblasts,miRNA-133 could promote the proliferation of myoblast.Finally,to further elucidate the mechanism of miRNA-1 and miRNA-133 on duck skeletal muscle development,the candidate target genes of miRNA-1 and miRNA-133 were identified through literature search and bioinformatics prediction.The expression of candidate target genes in myoblasts were detected by real-time qPCR after the transfection of miRNA mimics and inhibitors into duck myoblasts,and the results showed that miRNA-1 could significantly decrease the expression of HDAC4(P<0.01),and miRNA-133 could significantly decrease the expression of SRF and TGFBR1(P<0.01).Dual luciferase reporter system results revealed that miRNA-1 could inhibit the activity of pGL-Basic-HDAC4 luciferase reporter gene,while miRNA-133 did not decrease the activitives of pGL-Basic-SRF and pGL-Basic-TGFBRl fluorescence.Therefore,miRNA-1 can target HDAC4 to promote the differentiation of duck myoblasts,and miRNA-133 can affect the expression of SRF and TGFBR1 and promote the proliferation of duck myoblasts. |
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