序列变异对miR-378生物发生以及靶标关系的影响

Effects of Sequence Variation on the Biogenesis and Target Relationship of miR-378

本研究旨在探索序列变异对miR-378的结构、表达水平以及靶标关系的影响。利用PCR测序比对不同猪种miR-378的序列突变,预测突变对miR-378二级结构和自由能的改变;构建miR-378表达载体检测突变对其加工过程中各级产物表达水平的作用;运用TargetScan和TargetRank对突变引起的靶标关系变化进行分析;采用microRNA pull-down技术验证突变对靶标关系的影响。结果发现,在荣昌猪和内江猪群体内pre-miR-378的+49和+68位均发生A>G的序列变异,且改变了pre-miR-378的二级结构和自由能;这两个位点突变后影响了pri-miR-378到pre-miR-378的加工过程,提升了pre-miR-378和成熟体miR-378的表达水平;同时+49/A>G的突变改变了miR-378的功能及其靶标关系,其中GDF6和RAB10分别为突变后获得和缺失的靶基因,而Runx1t1、Galnt3为不受突变影响的靶基因。本研究的结果强调了miRNAs序列变异的重要性及其对miRNAs生物发生和功能的显著影响。

The aim of this study is to explore the effects of sequence variation on the structure,expression level and targets relationship of miR-378.PCR-sequencing was employed to align the sequence mutations of miR-378 in different pig breeds and to predict the changes of the secondary structure and free energy of miR-378 caused by the mutation.The miR-378 expression vectors was constructed to detect the effect of mutation on the expression level during the processing.TargetScan and TargetRank were used to analyze the change of target relationship caused by the mutation of miR-378.MicroRNA pull-down technique was adapted to verify the effect of mutation on the target relationship of miR-378.The results showed that there were two A>G mutations on the +49 and +68 position of pre-miR-378 in Rongchang and Neijiang pigs,and the secondary structure and free energy of pre-miR-378 were changed.And these two mutations affected the processing from pri-miR-378 to pre-miR-378,and enhanced the expressions of pre-miR-378 and mature miR-378.The mutation of +49/A>G changed the function and target relationship of miR-378,of which GDF6 and RAB10 were the target genes obtained and deleted after mutation,respectively,while the shared targets(Runx1t1 and Galnt3)were not affected by mutation.These results serve to highlight the importance of miRNA mutations and their impacts on miRNA biogenesis.