LncRNA13164 regulates immune response of Apis cerana cerana larvae to Ascosphaera apis infection via ace-miR-4968-y

Abstract:

[Objective] Long non-coding RNA (lncRNA) usually functions as competing endogenous RNA (ceRNA) to play crucial regulatory roles. As no study of the function of bee lncRNA is available, the role of lncRNA in the immune response of bee is unclear. This study aims to reveal the regulatory function and mechanism of lncRNA in immune response of Apis ceranacerana larvae to Ascosphaera apis infection. Through deep sequencing and bioinformatics analysis, we have found that lncRNA targets ace-miR-4968 and involves in the response of A. c. cerana larvae to A. apis infection. [Methods] Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was performed to quantify the expression of lncRNA13164 in larval guts of A. c. cerana after A. apis inoculation. ILoc-LncRNA was employed to predict subcellular localization of lncRNA13164. RNAhybrid, Miranda, and TargetScan were applied to predict target miRNAs of lncRNA13164 and miRNA-targeted mRNAs. PCR and RT-qPCR were performed to validate expression of lncRNA13164 and ace-miR-4968 as well as their potential binding relationship. The larvae which had been infected with A. apis were fed with dsRNA for RNAi of lncRNA13164 in larval guts, followed by determination of the silencing effect on lncRNA13164 and relative expression of ace-miR-4968 and three immune genes (stk, e3ul, and or1). [Results] The expression of lncRNA13164 was up-regulated in guts of 4-day-old larvae and significantly up-regulated in guts of 5- and 6-day-old larvae in the inoculation group as compared with that in the non-inoculation group. LncRNA13164 targeted 15 miRNAs including ace-miR-4968, which formed a regulatory network. ace-miR-4968 putatively targeted 79 genes which were involved in 17 gene ontology (GO) terms and 85 Kyoto encyclopedia of genes and genomes (KEGG) pathways. Both lncRNA13164 and ace-miR-4968 were expressed in A. c. cerana larval gut. The expression of lncRNA13164 in guts of both 5- and 6-day-old larvae was significantly down-regulated as compared with that in the dsRNA-egfp-fed group, and silencing efficiencies were 66.05% and 56.45%, respectively. After the silencing of lncRNA13164, the expression of ace-miR-4968 was up-regulated (P<0.01) in guts of 5-day-old larvae, while expression of stk, e3ul, and or1 was down-regulated (P<0.05). [Conclusion] lncRNA13164 in A. c. cerana larval guts can be silenced through the feeding of specific dsRNA. lncRNA13164 regulates the expression of serine/threonine-protein kinase Doa isoform X4 gene stk, E3 ubiquitin-protein ligase (MYLIP) gene e3ul, and oxidation resistance protein 1 isoform X6 gnen or1 via ace-miR-4968 and further mediates the immune response of host to A. apis invasion.