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捻转血矛线虫阿苯达唑耐药虫株给药前后miRNA差异表达谱及网络调控分析
王腾宇¹,陈昕迪¹,石雅琴¹,毛晓伟¹,闫旭¹,苏娅¹,温海峰¹,刘春霞²,王文龙^1*
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(1.内蒙古农业大学兽医学院/农业部动物疾病临床诊疗技术重点实验室, 呼和浩特 010018;2.内蒙古农业大学生命科学院, 呼和浩特 010018)

摘要:

旨在通过miRNA测序技术对捻转血矛线虫阿苯达唑给药前后的耐药虫株差异表达miRNA及网络调控分析，获取其转录本中全miRNA表达谱及耐药相关差异表达基因和miRNA，探究miRNA在捻转血矛线虫阿苯达唑耐药虫株给药前后的差异。采用Illumina Hiseq2 000平台对捻转血矛线虫阿苯达唑耐药虫株给药前和给药后的虫体进行Small RNA-seq测序，将Raw Data与参考基因组和miRBase数据库进行比对分析，使用RNAhybrid、Miranda和TargetScan对miRNA靶基因进行预测，筛选捻转血矛线虫耐药虫株给药前后表达量显著差异的miRNA并通过Stem-loop qRT-PCR验证，同时对差异显著的miRNA-mRNA调控关系采用Cytoscape软件进行可视化分析。将靶基因向KEGG和GO两大权威数据库映射，预测和分析靶基因功能注释情况和参与调控的通路情况。结果显示:1)捻转血矛线虫阿苯达唑耐药虫株给药前和给药后共筛选出显著差异表达的miRNA 188个，其中125个上调，63个下调，且qRT-PCR结果与转录组测序结果表达情况一致。2)显著差异表达的144个miRNA共关联到353个miRNA-mRNA负调控关系，注释到46条GO功能条目以及代谢，运输和降解等327条通路。3)筛选出的候选靶基因主要富集在ABC转运蛋白，HIF-1信号通路，cGMP - PKG 信号通路以及P450药物代谢通路，证实在药物胁迫的影响下捻转血矛线虫主要通过代谢和转运的方式参与耐药性调控。本研究为捻转血矛线虫耐药性诊断辅助分子标记的筛选和虫体耐药性调控因素及作用机制的研究提供参考。

关键词: 捻转血矛线虫阿苯达唑耐药性转录组 miRNA

DOI：10.11841/j.issn.1007-4333.2022.11.12

投稿时间：2021-08-16

基金项目:国家自然科学基金项目(31760731)；内蒙古自治区科技计划项目(201702074)

Analysis on the differential miRNA expression profiles and regulatory networks of albendazole-resistant strains of Haemonchus contortus before and after administration

WANG Tengyu¹,CHEN Xindi¹,SHI Yaqin¹,MAO Xiaowei¹,YAN Xu¹,SU Ya¹,WEN Haifeng¹,LIU Chunxia²,WANG Wenlong^1*

(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 010018, China;2.College of Life Science, Inner Mongolia Agricultural University, Hohhot 010018, China)

Abstract:

The aims of this study were to analyze the differentially expressed miRNAs and regulatory networks of Haemonchus contortus albendazole-resistant strains before and after albendazole administration by miRNA sequencing technology, to obtain the full miRNA expression profiles and drug resistance-related differentially expressed genes and miRNAs, and to explore the differences of DE miRNAs before and after Albendazole administration in H. contortus resistant strains. Small RNA-seq sequencing was performed on the Illumina Hiseq2000 platform for the pre- and post-administration of albendazole-resistant strains of H. contortus. The raw data were compared and analyzed with the reference genome and miRBase database. The miRNA target genes were predicted, and the miRNAs with significant differences in expression before and after administration of the transthyretin nematode resistant strains were screened and validated by stem-loop qRT-PCR. The miRNA-mRNA regulatory relationships with significant differences were visualized and analyzed using Cytoscape software. The target genes were mapped to KEGG and GO authoritative databases to predict and analyze the functional annotation of target genes and the pathways involved in regulation. The results showed that: 1)A total of 188 miRNAs with significantly differentially expression were screened before and after administration of albendazole-resistant strains of C. twistedus, of which 125 were up-regulated and 63 were down-regulated. The qRT-PCR results were consistent with that of transcriptome sequencing. 2)The 144 miRNAs significantly differentially expression were associated with 353 miRNA-mRNA negative regulatory relationships. Forty-six GO functions items and 327 pathways such as metabolism, transportation and degradation were annotated. 3)The screened candidate genes were mainly enriched in ABC transporter protein, HIF-1 signaling pathway, cGMP-PKG signaling pathway and P450 drug metabolism pathway, which confirmed the involvement of Haematobium twisted spear nematodes in drug resistance regulation mainly through metabolism and transport under the influence of drug stress. This study provides a reference for the screening of molecular markers for drug resistance diagnosis and the influencing factors and mechanisms of drug resistance regulation in H. contortus.

Key words: Haemonchus contortus albendazole drug resistance transcriptome miRNA