lincRNA_Tc13743.2-miR-133-5p-TcGSTm02 regulation pathway mediates cyflumetofen resistance in Tetranychus cinnabarinus

Differential expression of metabolic detoxification enzymes is an important mechanism involved in pesticide/ acaricide resistance of mite pests. The competing endogenous RNA hypothesis offers a new opportunity to investigate post-transcriptional regulation of those genes. In this study, 4454 long non-coding RNAs were identified in the carmine spider mite Tetranychus cinnabarinus by transcriptome sequencing. Software-based predictions indicated that a long intergenic non-coding RNA, (lincRNA)_Tc13743.2 and a detoxification enzyme gene, TcGSTm02, both contained a microRNA (miR-133-5p) response element. Over-expression of lincRNA_Tc13743.2 and TcGSTm02 were detected in a cyflumetofen-resistant T. cinnabarinus strain (CyR), whereas down-regulation of miR-133-5p was observed in this strain. Conversely, up-regulation of miR-133-5p could inhibit TcGSTm02 expression levels, and both lincRNA_Tc13743.2 and TcGSTm02 were significantly enriched in miR-133-5p biotin-avidin pull-down assays. RNA-binding protein immunoprecipitation assay showed that lincRNA_Tc13743.2 and TcGSTm02 bound to a silencing complex containing miR-133-5p. Moreover, a luciferase reporter assay based on a human cell line revealed that over-expression of lincRNA_Tc13743.2 could significantly reduce the inhibition exerted by miR-133-5p through the TcGSTm02 3 ' UTR. In addition, co -localization of lincRNA_Tc13743.2 and miR-133-5p was detected using fluorescence in situ hybridization, suggesting that lincRNA_Tc13743.2 interacts directly with miR-133-5p in spider mites. More importantly, silencing the expression of lincRNA_Tc13743.2 significantly reduced the expression levels of TcGSTm02 and increased the sensitivity of spider mites to cyflumetofen. Our data show that lincRNA_Tc13743.2 up-regulates TcGSTm02 expression by competing for miR-133-5p binding, demonstrating that a lincRNA_Tc13743.2-miR-1335p-TcGSTm02 pathway mediates cyflumetofen resistance in mites.