Screening Of Albendazole Resistance-associated MiRNA In Haemonchus Contortus And Its Regulatory Role

To reveal the possible regulatory mechanisms of non-coding RNAs in the development of resistance to albendazole in Haemonchus contortus,we selected standard sensitive strains and resistant strains before and after drug administration for Small RNA library sequencing analysis,subjected all known and new miRNAs to target gene prediction analysis,screened sensitive and resistant strains and resistant strains for significantly different miRNAs before and after drug administration.The miRNA-m RNA regulatory network was constructed,and the screened drug-resistant hco-miR-5896 and hco-miR-86 and their target genes WDFY3 and CAC were validated by a dual luciferase gene reporter system.The main findings are as follows:1.A total of 2955 miRNAs were identified by Small RNA sequencing analysis of standard sensitive and resistant strains of Albendazole.294 miRNAs were identified with FDR<0.05 and|log₂（Fold change）|≥1 as the screening condition for differential expression analysis,of which 113 were significantly up-regulated and 181 were significantly down-regulated.The differentially expressed miRNAs were functionally enriched and pathway annotated,and the differential miRNA target genes were mainly focused on GO terms such as metabolic pathways,macromolecular complexes and transport activities,as well as Fox O signaling pathway,m TOR signaling pathway and PI3K-Akt signaling pathway,all of which were related to drug resistance.2.Based on the Small RNA sequencing results before and after the administration of Albendazole-resistant strains of H.contortus,1455 and 1337 miRNAs were identified before and after the administration of the drug,respectively,and a total of 188 significantly differentially expressed miRNAs were screened,of which 125 were significantly up-regulated and 63 were significantly down-regulated.Functional enrichment analysis revealed that the significantly differentially expressed miRNA target genes were mainly involved in ATP binding and transport,metabolic processes and glycolysis,etc.The results of KEGG pathway enrichment showed that a large number of genes were annotated in ABC transporter protein,HIF-1 signaling pathway and cytochrome P450 drug metabolism pathway,which were consistent with the results of the previous transcriptome analysis.3.Based on the results of miRNA expression analysis,hco-miR-5896 and hco-miR-86,which had significant differences,were selected for target gene prediction by Target Scan,Miranda and RNAhybrid software,and the target fragments of WDFY3 and CAC were cloned and sequenced and bioinformatically analyzed,and these two genes were successfully constructed.The wild-type and mutant dual luciferase reporter gene vectors of these two genes were successfully constructed,and the target binding relationship between hco-miR-5896 and WDFY3 and hco-miR-86 and CAC was demonstrated by the dual luciferase reporter gene system.In summary,a large number of miRNAs have been identified through H.contortus albendazole-sensitive strains and drug-resistant strains,and two sets of regulatory miRNAs and target genes have been verified.The results of the study can provide a basis for the research on the resistance of H.contortus.