Comparative analyses of longissimus muscle miRNAomes reveal microRNAs associated with differential regulation of muscle fiber development between Tongcheng and Yorkshire pigs

Tongcheng (TC) and Yorkshire (YK) are two pig breeds with distinctive muscle morphology. Porcine microRNAome (miRNAome) of the longissimus muscle during five developmental stages (40, 55, 63, 70, and 90 days post coitum (dpc)) was explored by Solexa sequencing in the present study to find miRNAs involved in the different regulation of skeletal muscle development between the two breeds. A total of 320 known porcine miRNAs, 64 miRNAs corresponding to other mammals, and 224 potentially novel miRNAs were identified. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) suggested that the factor “pig breed” affected the miRNA expression profiles to a lesser extent than the factor “developmental stage”. Fifty-seven miRNAs were differentially expressed (DE) between the neighbor developmental stages in TC and 45 such miRNAs were found in YK, 34 in common; there were more down-regulated stage-DE miRNAs than up-regulated. And a total of 23, 30, 12, 6, and 30 breed-DE miRNAs between TC and YK were identified at 40, 55, 63, 70, and 90 dpc, respectively, which were mainly involved in cellular protein modification process, protein transport, and metabolic process. As the only highly expressed breed-DE miRNA found in no less than four developmental stages, and also a stage-DE miRNA found both in TC and YK, miR-499-5p could bind the 3’-UTR of a myofibrillogenesis regulator, destrin/actin depolymerizing factor (DSTN), as validated in dual luciferase reporter assay. The results suggested that miR-499-5p possibly play a noteworthy role in the breed-distinctive porcine muscle fiber development associated with the regulation of DSTN.

通城猪（Tongcheng, TC）与约克夏猪（Yorkshire, YK）是两种具有独特肌肉形态特征的猪品种。本研究采用Solexa测序技术，对两个品种背最长肌在五个发育阶段（妊娠后40、55、63、70和90天，days post coitum, dpc）的猪microRNA组（miRNAome）进行测序分析，以期筛选出参与调控两个品种骨骼肌发育差异的miRNA。本研究共鉴定得到320个已知猪源miRNA、64个对应其他哺乳动物的miRNA以及224个潜在新型miRNA。主成分分析（PCA）与层次聚类分析（HCA）结果显示，相较于发育阶段这一因素，猪品种因素对miRNA表达谱的影响程度更低。通城猪相邻发育阶段间存在57个差异表达（differentially expressed, DE）miRNA，约克夏猪则为45个，其中二者共有34个差异表达miRNA；且阶段特异性差异表达miRNA中，下调表达的数量多于上调表达的。在妊娠后40、55、63、70及90 dpc五个时间点，分别鉴定得到23、30、12、6和30个通城猪与约克夏猪间的品种特异性差异表达miRNA，这些miRNA主要参与细胞蛋白质修饰过程、蛋白质转运及代谢过程。miR-499-5p是唯一一个在至少四个发育阶段中高表达的品种特异性差异表达miRNA，同时也是通城猪与约克夏猪中均存在的阶段特异性差异表达miRNA；双荧光素酶报告基因实验验证表明，该miRNA可结合肌纤维发生调节因子丝切蛋白/肌动蛋白解聚因子（destrin/actin depolymerizing factor, DSTN）的3’-UTR区域。研究结果提示，miR-499-5p可能通过调控DSTN的表达，在品种特异性的猪肌纤维发育过程中发挥显著作用。