| 摘要: |
| 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5 374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1 320个上下游基因，共匹配1 566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3 227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。 |
| 关键词: 捻转血矛线虫 伊维菌素 耐药性 长链非编码RNA 竞争性内源RNA |
| DOI：10.11841/j.issn.1007-4333.2023.01.16 |
| 投稿时间：2022-05-07 |
| 基金项目:内蒙古自然科学基金项目(2020MS03012)；内蒙古自治区科技计划项目(201702074) |
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| Analysis of long non-coding RNAs associated with Ivermectin resistance and its regulatory function in Haemonchus contortus |
|
CHEN Xindi1,WANG Tengyu1,LIU Chunxia2,WANG Wenlong1*
|
| (1.Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Disease of Ministry of Agriculture/College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010010, China;2.College of Life Science, Inner Mongolia Agricultural University, Hohhot 010010, China) |
| Abstract: |
| 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)5 374 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 1 320 upstream and downstream genes through cis-regulation, matching a total of 1 566 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 3 227 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. |
| Key words: Haemonchus contortus Ivermectin drug resistance long non-coding RNA competing endogenous RNA |
