猪miR-23a-27a-24簇启动子预测及活性分析

Prediction and activity analysis of the promoter of the porcine miR-23a-27a-24 cluster

目的 本研究旨在对猪miR-23a-27a-24簇(miR-23a-27a-24 cluster)的潜在启动子区域进行预测和分析,为进一步探索miR-23a-27a-24基因对猪转录调控机制的研究奠定基础。方法 利于生物信息学分析猪miR-23a-27a-24簇的启动子区域、CpG岛及其潜在的转录因子;利用截短突变和PCR技术克隆得到5个逐级缺失的miR-23a-27a-24基因启动子片段,构建双荧光素酶报告载体,通过检测各载体的双荧光素酶活性获得miR-23a-27a-24基因的核心启动子区域。结果 pGL3-cMiR启动子在猪miR-23a-27a-24簇上游341bp至994bp区荧光素酶活性最强,为pGL3-basic空载体活性的5.02倍(P<0.05),视为miR-23a-27a-24簇核心启动区。生物信息学分析表明:猪miR-23a-27a-24簇启动子区不含CpG岛,其核心启动子区可能存在SP1、VDR、MAZ、YY1、E2F1和ELF1六种潜在的转录因子结合位点。结论 本研究构建并确定了猪miR-23a-27a-24簇核心启动子区域,可为今后开展猪miR-23a-27a-24簇的转录调控机制奠定基础。

Objective The aim of this study was to predict and analyse the potential promoter regions of the porcine miR-23a-27a-24 cluster and to lay the foundation for further exploration of the function of the miR-23a-27a-24 gene on the transcriptional regulatery mechanism in pigs.Methods The promoter region of the porcine miR-23a-27a-24 cluster,the CpG island and its potential transcription factors were analyzed by bioinformatics;five promoter fragments of the miR-23a-27a-24 gene with stepwise deletion were cloned by using truncated mutation and PCR techniques,and a dual luciferase reporter vector was constructed,and the dual luciferase activity of each vector was assayed to obtain the miR-23a-The core promoter region of the miR-23a-27a-24 gene was obtained by assaying the dual luciferase activity of each vector.Results showed that the pGL3-cMiR promoter had the strongest luciferase activity in the 341bp to 994bp region upstream of the porcine miR-23a-27a-24 cluster,which was 5.02 times higher than that of the pGL3-basic empty vector(P0.05),and was regarded as the core promoter region of the miR-23a-27a-24 cluster.Bioinformatics analysis showed that the promoter region of porcine miR-23a-27a-24 cluster did not contain CpG islands,and six potential transcription factor binding sites,SP1,VDR,MAZ,YY1,E2F1 and ELF1,might exist in its core promoter region.Conclusions This study constructed and identified the core promoter region of the porcine miR-23a-27a-24 cluster,which can lay the foundation for future work on the transcriptional regulatory mechanism of the porcine miR-23a-27a-24 cluster.