Zhang Wei, Wang Limin, Zhou Ping, Song Guangchao, Shen Min, Gan Shangquan, Shi Guoqing
College of Animal Science and Technology, Shihezi University, Shihezi, 832003, China; Institute of Animal Husbandry and Veterinary Medicine, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi 832000, China.
Institute of Animal Husbandry and Veterinary Medicine, Xinjiang Academy of Agricultural and Reclamation Sciences, Shihezi 832000, China.
PLoS One. 2015 Feb 20;10(2):e0117327. doi: 10.1371/journal.pone.0117327. eCollection 2015.
MicroRNAs (miRNAs) are key regulators in miRNA-mediated gene regulatory networks and play important roles in many biological processes, such as growth and development of mammals. In this study, we used microarrays to detect 261 miRNAs that are expressed in sheep skeletal muscle. We found 22 miRNAs that showed high levels of expression and equated to 89% of the total miRNA. Genetic variations in these 22 pri-miRNAs were further investigated using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing. A total of 49 genetic variations, which included 41 single nucleotide polymorphisms (SNPs) and 8 deletions/insertions, were identified in four sheep breeds. Three variations were further researched in a larger sample set, including five sheep breeds with different meat production performances. We found that the genotype and allele frequencies of the CCC deletion/insertion in pri-miR-133a were significantly related to the sheep meat production trait. Finally, cell assays and quantitative reverse transcription PCR (qRT-PCR) were employed to investigate the effect of pri-miRNA genetic variation on the miRNA biogenesis process. The results confirmed that genetic variations can influence miRNA biogenesis and increase or decrease the levels of mature miRNAs, in accordance with the energy and stability change of hair-pin secondary structures. Our findings will help to further the understanding of the functions of genetic variations in sheep pri-miRNAs in skeletal muscle growth and development.
微小RNA（miRNA）是miRNA介导的基因调控网络中的关键调节因子，在许多生物学过程中发挥重要作用，如哺乳动物的生长发育。在本研究中，我们使用微阵列检测了在绵羊骨骼肌中表达的261种miRNA。我们发现22种miRNA表达水平较高，占总miRNA的89%。使用聚合酶链反应-单链构象多态性（PCR-SSCP）和测序进一步研究了这22种初级miRNA（pri-miRNA）的遗传变异。在四个绵羊品种中总共鉴定出49个遗传变异，其中包括41个单核苷酸多态性（SNP）和8个缺失/插入。在一个更大的样本集中对三个变异进行了进一步研究，该样本集包括五个具有不同产肉性能的绵羊品种。我们发现pri-miR-133a中CCC缺失/插入的基因型和等位基因频率与绵羊肉生产性状显著相关。最后，采用细胞试验和定量逆转录PCR（qRT-PCR）研究pri-miRNA遗传变异对miRNA生物合成过程的影响。结果证实，遗传变异可影响miRNA生物合成，并根据发夹二级结构的能量和稳定性变化增加或降低成熟miRNA的水平。我们的研究结果将有助于进一步了解绵羊pri-miRNA的遗传变异在骨骼肌生长发育中的功能。
