HLA—DQA1 3’-UTR序列多态性分析及microRNA的调控

Polymorphism analysis of 3＇-UTR in HLA-DQA1 and microRNA regulation

目的揭示HLA-DQA1基因3’-UTR区多态性的进化机制及其对基因表达的调控功能。方法利用核苷酸变异率计算、系统发育树构建等群体遗传学分析方法将IMGT／HLA数据库中释放的20条HLA-DQAl全长序列多态性进行系统分析，揭示3’-UTR多态性的进化机制。利用mirandav1．9软件提供的算法，对HLA-DQAl三种差异较大的3’UTR等位基因序列进行microRNA的靶位点预测，对分值较高的预测利用体外荧光细胞实验进行验证。结果HLA—DQAl基因的3’-UTR具有极高的核苷酸变异率及跨物种多态性，表明其受到平衡选择的作用。预测出14种分值较高的microRNA-靶标复合物，且14个micfoRNA与3种DQA1-3’-UTR等位基因靶标的结合自由能以及分值均有不同程度的差异。通过体外荧光表达实验证明，HLA—DQAl受到hsa-mir-658的负调控作用，且hsa—mir-658对DQA1—3’-UTR3种等位基因特异性的结合对基因表达产生了影响。结论HLA—DQA1基因表达水平受到microRNA的负调控，且HLA—DQA13’-UTR的多态性可影响与microRNA的结合而引起等位基因特异性表达。

Objective To disclose the evolution mechanism of polymorphisms in HLA-DQA1 3＇UTR and their function on gene expression regulation. Methods The methods to study the population genetics, such as the nueleotide diversity computation, the phylogenetie tree construction, etc. , are used to analyze the polymorphism of 20 genomic full length sequences of HLA-DQA1, which are released in IMGT/HLA database. Miranda vl. 9 is used to predict microRNA-targets for 3 different alleles of DQA1-3＇ -UTR. Top predictions are confirmed by Luciferase reporter analysis in vitro. Results We find high level of nucleotide diversity and trans-species polymorphism in DQA1-3＇ -UTR, suggesting balancing selection on this region. Fourteen top pre- dictions of microRNA-targets are obtained. The score and free energy of microRNA-targets among 3 alleles are different. Luciferase reporter analysis indicates that HLA-DQA1 is under negative control by hsa-mir-658, and the allele-specific binding to microRNA influences the expression level of luciferase. Conclusions The expression of HLA-DQA1 is under negative regulation by microRNA. Moreover, the polymorphism of DQA1 3＇ -UTR may influence the binding ability with microRNA and produce the allele-specific expression.