Differential Expressed Analysis And Functional Study Of MicroRNAs After Infection Of BMBC By Echinococcus Granulosus

Echinococcus granulosus?E.granulosus?is a serious global zoonotic parasitic pathogen that seriously endangers human health and animal husbandry.In recent years,microRNAs?miRNAs?have become one of the research hotspots because they play an important role in the regulation of gene expression.Studies have shown that miRNAs play a crucial role in parasite-host interactions,and the regulation effect of miRNA can help parasites escape the host?s immune response.Therefore,in this study,high-throughput transcriptome sequencing was used to construct miRNA expression profiles at different time points of mouse bone myeloid dendritic cells?BMDC?and protoscoleces?PSC?after PSC infected BMDC.Screening differentially expressed miRNAs,predicting target genes,analyzing the cellular pathways and functions of differentially expressed miRNAs target genes,and screening immune-related miRNAs in BMDCs and immune evasion-related miRNAs in PSCs.This study explored the interaction between PSC and host cells from the perspective of miRNAs and provided a new idea for revealing the pathogenesis of E.granulosus.It also provided a new perspective on the prevention and control of E.granulosus.Purposes:?1?Construction of miRNA libraries of PSC and BMDC after PSC infected BMDC;?2?To Analyze the pathway and cellular processes of enrichment of differentially expressed miRNA target genes,studying the mechanism of miRNAs in E.granulosus-host interaction,and to screen immune response-related miRNAs in BMDCs and immune evasion-related miRNAs in PSCs.Laying the foundation for the study of the pathogenesis of E.granulosus at the cellular level;?3?Screening an immune-related miRNA from BMDC differentially expressed miRNAs,preliminary study its function.Methods:?1?Samples were taken at 6 h,12 h,and 24 h after PSC infected BMDC,respectively.12 total RNA samples were extracted: PX₆?BMDC in the 6-hour infected group?,PX₁2,PX₂4,PBS₆?BMDC in the 6-hour control group?,PBS₁2,PBS₂4,PSC₆?PSC in the 6-hour infected group?,PSC₁2,PSC₂4,E.granulosus₆?PSC in the 6-hour control group?,E.granulosus₁2 and E.granulosus₂4.Qualified samples were used to construct miRNA libraries,sequencing on an Illumina HiSeqTM 2500/MiSeq sequencing platform,raw data was evaluated and filtered to obtain high quality reads.?2?To screen differentially expressed miRNAs with qvalue<0.01 and |log2?foldchange?|>1 as the cut-off values,and predicting target genes of differentially expressed miRNAs by bioinformatics methods,using GOseq R software package and KOBAS software to conduct enrichment analysis on the predicted target genes.To screen differentially expressed miRNAs related to immune evasion-related in PSC libraries and immune responses in BMDC libraries.Randomly selected differentially expressed miRNAs and verified the consistent with high-throughput sequencing results by qPCR.?3?The immune response-related mmu-miR-301a-5p in the BMDC libraries was screened by bioinformatics analysis.Whether the mmu-miR-301a-5p binds to the mRNA 3?-UTR of the predicted target gene Heat shock 70 kDa protein 4?Hspa4?was detected by the dual luciferase reporter gene.The chemically synthesized mmu-miR-301a-5p mimics,mimics negative control?NC?,inhibitor and inhibitor NC were transfected into BMDC,and then qPCR were used to detect the expression of Hspa4 in each group,thereby verifying the regulatory relationship between mmu-miR-301a-5p and Hspa4.PSC was used to infect BMDC that has been transfected mimics,and detecting cytokine content by ELISA.Results:?1?6 miRNA libraries of BMDC?3 for the infection group and 3 for the control group?and 6 miRNA libraries of PSC were established.A total of 3.777 Gb data and 72,489,485 reads were obtained from 6 libraries of BMDC.The Q20 and Q30 of each library in the quality analysis were higher than 98% and 97% respectively,and the error rate was 0.01%.A total of 3.536 Gb of data and 70,748,082 reads were measured in the 6 libraries of PSC.The Q20 and Q30 of each library were higher than 98% and 96% respectively,and the error rate was 0.01%.The library quality assessment was qualified and met the requirements for subsequent research;after data filtering,a total of 140,455,720 high-quality reads were obtained.?2?Compared with the control group,30,34 and 32 miRNAs were differentially expressed in PSC and 9,16 and 58 miRNAs were differentially expressed in BMDC.GO results showed that differentially expression miRNA predicted target gene enrichment in PSC in biological functions,such as protein collection.Target genes predicted by differential miRNA in BMDC were mainly enriched in cell group classification,such as membrane organelles.KEGG analysis revealed that PSC differentially expressed miRNAs predicted that target genes were enriched in pathways such as the mitogen-activated protein kinase signaling pathway?MAPK signaling pathway?;BMDC differentially expressed miRNAs predicted that target genes were mainly enriched in NF-kappa B signaling pathway.In PSC libraries,immune-evasive-related miRNAs were egr-miR-2p-5p,egr-miR-219,egr-miR-71,etc.,while in BMDC libraries,immune-related miRNAs were mmu-miR-3074-2-3p,mmu-miR-193a-3p,mmu-miR-301a-5p,etc.?3?The results of dual luciferase assay showed that luciferase activity was significantly lower in the mimics group compared with the inhibitor group,the inhibitor NC group,and the mimics NC group?P<0.05?.After mimics were transfected into BMDC,the expression of Hspa4 was detected by qPCR.Comparing with other groups,the expression of Hspa4 gene in mimics group was significantly lower?P<0.05?.Mmu-miR-301a-5p promoted the secretion of Th2 type cytokines IL-4 and IL-10 in the early stage of PSC infection of BMDC?24 h?.Conclusions:?1?The miRNA library of PSC infected BMDC at different time points was successfully constructed,and the library quality evaluation was qualified,which met the requirements for subsequent research.?2?During the process of PSC infection,the expression of miRNA in PSC and BMDC both changed rapidly and significantly,especially the immune-evading related miRNA in PSC and the immune-responsive related miRNA in BMDC,suggesting that miRNA may play an important regulatory role in E.granulosus-host interaction.?3?Hspa4 gene is one of the mmu-miR-301a-5p target genes.It is speculated that mmu-miR-301a-5p was beneficial to the immune evasion and persistent infection of E.granulosus in the early stage of PSC infected BMDC?24 h?.