A comprehensive microRNA expression profile related to hypoxic adaptation in Tibetan pig. Sus scrofa

In this study, miRNA-seq technique was used to identify differentially expressed miRNAs (DE miRNAs) in cardiac muscle of the Tibetan pig (TP) and Yorkshire pig (YP), which were both raised in highland environments. We obtained 108 M clean reads and 372 unique miRNAs that included 210 known pre-miRNAs and 162 novel pre-miRNAs. In addition, 20 DE miRNAs, including 10 upregulated and 10 downregulated miRNAs, were identified by comparing TP and YP. Based on the expression abundance and differentiation between the two populations, we predicted their targets, and KEGG pathway analyses suggested that DE miRNAs between the Tibetan pigs and Yorkshire pigs are involved in hypoxia-related pathways, such as the MAPK, mTOR, and VEGF signaling pathways, cancer-related signaling pathways, etc. Five DE miRNAs were randomly selected to validate the veracity of miRNA-seq using real-time PCR. The results showed that the expression corresponds to the trend in miRNA-seq, hence the deep-sequencing methods were feasible and efficient. This study expanded the number of hypoxic-adaptation-related miRNAs in pig and indicated that the expression patterns of hypoxia-related miRNAs are significantly altered in the Tibetan pig. DE miRNAs may play important roles in hypoxic adaptation after migration to hypoxic environments. Overall design: mRNA profiles of 6-month old Tibetan pig (TP) and Yorkshire pig (YP) were generated by deep sequencing, in duplicate, using Hiseq 2000.

本研究采用miRNA测序（miRNA-seq）技术，对高原环境下饲养的藏猪（Tibetan pig, TP）与约克夏猪（Yorkshire pig, YP）的心肌组织中差异表达miRNA（differentially expressed miRNAs, DE miRNAs）进行鉴定。本研究共获得108百万条clean reads，以及372个独特miRNA，其中包含210个已知前体miRNA与162个新型前体miRNA。此外，通过对比藏猪与约克夏猪的转录组数据，共鉴定出20个差异表达miRNA，其中10个表达上调、10个表达下调。基于两个猪群的表达丰度与表达差异，我们对其靶基因进行了预测；京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路分析结果显示，藏猪与约克夏猪之间的差异表达miRNA参与了低氧相关通路，如丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）、哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）以及血管内皮生长因子（vascular endothelial growth factor, VEGF）信号通路，亦涵盖癌症相关信号通路等。本研究随机选取5个差异表达miRNA，采用实时荧光定量PCR（real-time PCR）验证miRNA-seq结果的准确性，结果显示其表达趋势与miRNA-seq分析结果一致，证实了深度测序方法的可行性与高效性。本研究扩充了猪体内低氧适应相关miRNA的数量，并表明低氧相关miRNA的表达模式在藏猪中发生了显著改变。差异表达miRNA可能在猪迁移至低氧环境后的低氧适应过程中发挥重要作用。整体实验设计：采用HiSeq 2000平台对6月龄藏猪与约克夏猪的心肌组织mRNA进行深度测序，每组设置两个生物学重复。