Construction Of Differentially Expressed MRNA And NcRNA And Their Roles In Artemisinin-resistant Plasmodium Falciparum

Objective:Artemisinin resistance is associated with K13 mutation,but the resistance mechanism is still not clear.Resistant and normal falciparum malaria RNA-seq data were downloaded from the GEO database and analyzed using bioinformatic and statistical methods to explore its possible biological functions and pathways in the resistance mechanism.We also try to verify the result by experiments in vitro.Methods:The gene expression profiles GSE119231 and GSE133236 were downloaded from the GEO database,and the online website Network Analyst was used to analyze the data and screen differentially expressed genes.Visualization of GO functional analysis,KEGG,Reactome and Pathview pathway enrichment analysis were performed on the differential m RNAs above.Protein-Protein Interaction Networks(PPI)of differentially expressed m RNAs were constructed from the STRING database,and Cytoscape software was used to visualize the network and screen hub-genes.A reference gene set was constructed and gene set enrichment analysis was performed on m RNA expression data using GSEA software.We also annotated the differentially expressed nc RNAs and searched their interaction in lnc RNA databases.We resuscitated Plasmodium falciparum 3D7 and 3D7C580 Y knockout strains in vitro,and detected the resistance of the strains by ring stage survival assay after early ring synchronization and late schizont synchronization.Results:From the normal NF54 and resistant PB58 groups,98 different expressed m RNAs were identified in ring stage,56 in trophozoite stage and 1 in schizont stage.The result of GO analysis was enriched in gene functions such as adhesion retention and material transport.For KEGG,Reactome and Pathview pathway enrichment analysis,the malaria pathway was enriched in both ring stage and trophozoite stage.Other significant enrichment pathways in the ring stage include lipid metabolism pathways(fatty acid biosynthesis,fatty acid degradation,fatty acyl-Co A biosynthesis,etc.)and peroxisome pathway.While the non-specific serine/threonine protein kinase and RNA degradation pathway were concentrated in trophozoite.Through PPI analysis,the interaction network of differentially expressed m RNAs in the ring stage and trophozoite stage was determined,and the top six hub-genes were screened out.GSEA analysis showed that mismatch repair,spliceosome and ructose and mannose metabolism pathways were enriched in trophozoite stage of PB58 whlie cysteine and methionine metabolism was enriched in trophozoite stage of NF54.In schizont stage,glycolysis/gluconeogenesis,valine,leucine and isoleucine degradation and SNARE interactions in vesicular transport were enriched in PB58.Four differentially expressed nc RNAs were screened in ring stage,and two in trophozoite stage,including three sn RNAs,one lnc RNA(telomerase RNA),and two unannotated nc RNAs(PF3D7＿0731000,PF3D7＿0918500).In vitro experiment results showed that 3D7 and 3D7 C580 Y knockout strains were morphologically similar in red blood cell stage.In ring stage survival assay,the survival rate of 3D7 and 3D7 C580 Y knockout strains were both <1%.Conclusion:1.The differentially expressed genes between normal NF54 strains and PB58 resistant strains were mainly distributed in the ring stage and trophozoite stage.K13 mutations may participate in the mechanism of resistance through the adhesion retention pathway represented by Pf EMP1,fatty acid metabolism pathway represented by longchain acyl-Co A synthetase,transport pathway represented by Q8IFL7,genetic information processing pathways such as mismatch repair,spliceosome and RNA degradation represented by LRP1 and various sn RNA and glycan and amino acid metabolism five aspects.2.In addition to sn RNA,telomerase RNA and unannotated nc RNA PF3D7＿0731000and PF3D7＿0108900 were differently enriched,which is worthy for further research and verification.