捻转血矛线虫伊维菌素耐药相关长链非编码RNA及其调控功能分析

Analysis of long non-coding RNAs associated with Ivermectin resistance and its regulatory function in Haemonchus contortus

为探究捻转血矛线虫伊维菌素耐药分子标记和关键调控元件,通过高质量的长链非编码RNA(Long non-coding RNA,lncRNA)表达谱对捻转血矛线虫敏感与耐药虫株lncRNA的顺式调控(Cis-regulation,cis)和竞争性内源RNA(Competing endogenous RNA,ceRNA)调控进行分析,筛选影响虫体伊维菌素耐药机制的微小调控分子。本研究利用RNA-seq技术对捻转血矛线虫伊维菌素敏感虫株和耐药虫株进行测序,根据生物学软件预测结果,筛选出位于蛋白编码基因上下游10 kb以内的lncRNA作为顺式调控作用的元件。对差异lncRNA进行GO和KEGG富集分析,并将显著差异的lncRNA和miRNA通过Target finder和Cytoscape v3.7.1软件建立lncRNA-miRNA调控网络和可视化分析。根据miRNA调控元件对lncRNA-circRNA-miRNA-mRNA调控网络进行连通性分析,结合耐药相关信号通路的富集结果构建lncRNA-miRNA-mRNA可视化网络,同时对随机选取的10个差异lncRNA进行qRT-PCR验证。结果显示:1)捻转血矛线虫伊维菌素敏感虫株和耐药虫株中筛选出5374条转录本,共预测到858个新lncRNA中有474个靶向到mRNA,且有205个lncRNA差异表达显著。其中,102个上调、103个下调。预测结果中723个lncRNA通过顺式调控作用靶向到1320个上下游基因,共匹配1566组调控关系。2)GO和KEGG富集结果显示,大量靶基因涉及到催化活性、转运活动、代谢过程、细胞膜以及代谢途径、内吞作用、甘油磷脂代谢、转运等信号通路。3)根据lncRNA-miRNA调控网络分析显示,205个显著差异lncRNA靶向结合371个miRNA,进而组成3227组调控关系且连通性较高。4)qRT-PCR验证结果显示,10个差异表达的lncRNA与RNA-seq结果表达趋势一致。综上,该研究成功获取lncRNA表达谱并构建部分耐药相关通路的lncRNA-miRNA-mRNA调控网络,为进一步探究lncRNA在捻转血矛线虫伊维菌素耐药调节机制中的作用提供理论依据。

To explore molecular markers and key regulatory elements of ivermectin resistance in Haemonchus contortus,the cis-regulation(cis)and competing endogenous RNA(ceRNA)regulation of lncRNA in H.contortus sensitive and resistant strains by high-quality long non-coding RNA(lncRNA)expression profiling,and the micro-regulatory molecules affecting the mechanism of ivermectin resistance were screened.In this study,RNA-seq technique was used to sequence the ivermectin-sensitive and drug-resistant strains of H.contortus.According to the prediction results of biological software,lncRNAs located in the upstream and downstream of the protein coding genes 10 kb were selected as the cis-regulatory element.The enrichment of differential lncRNAs were analyzed by GO and KEGG,and the significantly different lncRNAs and miRNAs were established by Target Finder and Cytoscape v3.7.1 software to establish lncRNA-miRNA regulation network and visual processing.According to the miRNA regulatory elements,the connectivity of the lncRNA-circRNA-miRNA-mRNA regulatory network was analyzed,and combined with the enrichment results of drug resistance-related signal pathways,the lncRNA-miRNA-mRNA visualization network was constructed.At the same time,ten randomly selected differential lncRNAs were verified by qRT-PCR.The results showed that:1)5374 transcripts of the sensitive and resistant strains of H.contortus predicted 858 new lncRNAs and targeted 474 mRNAs,of which 102 were up-regulated and 103 down-regulated.In the predicted results,723 lncRNAs were targeted to 1320 upstream and downstream genes through cis-regulation,matching a total of 1566 regulatory relationships.2)GO and KEGG enrichment results showed that a large number of target genes were involved in catalytic activity,transport activity,metabolic process,cell membrane and metabolic pathway,endocytosis,glycerol phospholipid metabolism,transport and other signal pathways.3)According to the analysis of lncRNA-miRNA regulatory network,205 significantly differential lncRNAs targeted and combined with 371 miRNAs to form 3227 groups of regulatory relationships with high connectivity.4)The results of qRT-PCR verification showed that the expression trend of 10 differentially expressed lncRNAs were consistent with that of RNA-seq.In summary,this study successfully obtained the lncRNA expression profile and constructed the lncRNA-miRNA-mRNA regulatory network of some drug resistance-related pathways,which provides a theoretical basis for further exploring the role of lncRNA in the regulation mechanism of ivermectin resistance in H.contortus.