猪miR-192/-215的组织表达谱及其关键靶基因分析

Tissue Expression Profile and Key Target Genes Analysis of Porcine miR-192 and miR-215

【目的】在前期通过Illumina Solexa高通量测序技术并结合荧光定量验证筛选出断奶仔猪E.coliF18菌株感染下表达量极显著上调的micro RNAs—miR-192和miR-215的基础上,为了解miR-192和miR-215的组织表达情况,进一步筛选确定miR-192和miR-215靶向调控E.coli F18菌株感染的关键靶基因,为探讨miR-192和miR-215调控断奶仔猪E.coli F18感染的分子调控机制奠定基础。【方法】试验采用Real-time PCR方法检测miR-192和miR-215在35日龄梅山仔猪各个组织中的分布情况,同时利用MEGA 5.0分析了其保守性,Target Scan预测miR-192和miR-215的靶基因,并对靶基因分别进行Gene Ontology和Pathway通路的富集分析,进一步利用DAVID对靶基因蛋白进行功能分类,最后将预测的靶基因与课题组前期筛选出的断奶仔猪E.coli F18感染抗性相关的调控基因取交集。【结果】miR-192和miR-215在35日龄梅山仔猪十二指肠和空肠中均高度表达,二者成熟序列在脊椎动物中高度保守。miR-192和miR-215对应的靶基因相同,具有156个靶基因,只在轴突导向通路(axon guidance pathway)显著富集(P-value<0.05)以及25个GO功能中显著富集(P-value<0.05)。靶基因蛋白按功能分为12类,其中信号转导功能的磷蛋白(phosphoprotein)和DNA结合蛋白(dna-binding)占主要部分。靶基因与前期筛选的E.coli F18感染抗性相关的调控基因(GEO登录号:GSE26854)取交集,发现DLG5、ALCAM、FRMD4B、MIPOL1和ZFHX3等5个基因为miR-192和miR-215的重要靶基因,其中DLG5为维持小肠上皮细胞结构完整性的重要因子。【结论】miR-192与miR-215是参与维持断奶仔猪肠道正常功能与抗F18大肠杆菌感染的重要因子,DLG5可能是miR-192和miR-215靶向调控E.coli F18菌株感染中关键的靶基因,今后需要进一步验证其能否作为梅山猪抗F18大肠杆菌的有效遗传标记。

【Objective】The previous study made by the authors＇ group had screened out two micro RNAs with extremely significantly increased expression for E.coli F18 infection：mi R-192 and mi R-215,using Illumina Solexa high-throughput sequencing technology combined with fluorescence quantitative determination.The objective of this study is to understand the mi R-192/ mi R-215 expression and further screen out the key target genes of mi R-192 and mi R-215 regulating E.coli F18 infection,which will lay a foundation for probing into the molecular mechanisms of regulating E.coli F18 infection in weaned piglets.【Method】Real-time PCR was used to detect the mi R-192/mi R-215 expression in different tissues of Meishan piglets at 35 days of age,meanwhile conservative sequence was analyzed using MEGA 5.0 software.The algorithms Target Scan was used to predict mi R-192/mi R-215 putative target genes,whose enrichment of Gene Ontology and Pathway was analyzed,respectively.Then the functional classification of target protein was further conducted using DAVID,finally,the intersection between predicted target genes and regulatory genes was taken for the resistance to E.coli F18 infection of weaned piglets which were screened out by the previous study.【Result】 The results indicated that mi R-192/mi R-215 were highly expressed in the duodenum and jejunum tissues of Meishan piglets at 35 days of age.The mature sequences of mi R-192 and mi R-215 are highly conservative in vertebrates.A total of 156 target genes of mi R-192/mi R-215 were exactly the same,which were significant enrichment in Axon guidance pathway and in 25 GO function（P〈0.05）.Target proteins were divided into 12 categories according to the function,and most of the target proteins belonged to phosphoprotein with signal transduction function and DNA binding protein.Through intersection between target genes and regulatory genes（Gene Expression Omnibus,Accession number：GSE26854） for the resistance to E.coli F18 infection,five important target genes such as ALCAM,DLG5,FRMD4 B,MIPOL1 and ZFHX3 were attained.From the point of target genes function,DLG5 was an important factor for maintaining epithelial cells structural integrity.【Conclusion】The results of this study revealed that mi R-192 and mi R-215 were two important factors of participating in maintaining the intestines function and resisting the E.coli F18 infection,DLG5 was likely to be the important target gene of mi R-192 and mi R-215 regulating E.coli F18 infection,and further analyses should be conducted to testify whether the DLG5 could be an effective genetic marker for the resistance to E.coli F18 infection in Chinese native pig breeds.