Abstract:

Objective To analyze and identify the expression profiles of oxidative stress-related genes and circular RNAs （circRNAs） in ricin toxin （RT）-induced pyroptosis of mouse mononuclear macrophages （RAW264.7） using transcriptome sequencing and bioinformatics technology， and to preliminarily analyze their potential functions. Methods The macrophages （RAW264.7 cells） were treated with RT to establish a cell pyroptosis model and divided into control group， 40 μg·L^-1 RT group， and 80 ng/mL RT group. Transmission electron microscope （TEM） was used to observe the morphology of the RAW264.7 cells in various groups； Western blotting method was used to detect the expression levels of the pyroptosis pathway-related proteins in the cells in various groups； 80 μg·L^-1 RT was selected for subsequent experiments. Transcriptome sequencing （RNA-Seq） was performed to obtain the circRNA and mRNA expression profiles in the RAW264.7 cells in control group and RT-treated group， followed by bioinformatics analysis. Results Compared with control group， the cells in 40 and 80 μg·L^-1 RT groups underwent morphological changes； the cells in RT groups showed obvious pyroptosis-like morphological changes， characterized by significant swelling of dying cells and the appearance of characteristic large bubbles on the plasma membrane. Compared with control group， the expression level of gasdermin DN-terminal fragment （GSDMD-N） protein in 40 and 80 μg·L^-1 RT groups was increased （P<0.05）； compared with 40 μg·L^-1 RT group， the expression level of GSDMD-N protein in the cells in 80 μg·L^-1 RT group was increased （P<0.05）； therefore， the subsequent experiments used the RT concentration of 80 μg·L^-1. A total of 2930 differentially expressed messenger RNAs （mRNAs） and 24 differentially expressed circRNAs were identified. The constructed circRNA-microRNA （miRNA）-mRNA competing endogenous RNA （ceRNA） regulatory network consisted of 7 circRNAs， 12 miRNAs and 13 mRNAs. Gene Ontology （GO） functional enrichment analysis showed that in biological process （BP）， the functions regulated by differentially expressed genes mainly included immune response and oxidative stress response； in cellular component （CC）， differentially expressed genes were mainly localized to the external side of plasma membrane and cell leading edge； in molecular function （MF）， they were mainly involved in transporter transmembrane activity and hormone receptor binding. Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis showed that differentially expressed genes were mainly enriched in Toll-like receptor signaling pathway， Forkhead box O（FoxO） signaling pathway and nuclear factor kappa-light-chain-enhancer of activated B cells （NF-κB） signaling pathway. In the protein-protein interaction （PPI） network， the top 10 hub genes with the highest connectivity were screened by CytoHubba， including matrix metalloproteinase 9 （MMP9）， superoxide dismutase 2 （SOD2）， and v-src sarcoma viral oncogene homolog （Src）. Conclusion The expression profiles of oxidative stress-related genes and circRNAs in RAW264.7 cells are altered after RT treatment. The screened differentially expressed circRNAs and mRNAs may serve as potential targets to regulate RT-induced pyroptosis in RAW264.7 cells through oxidative stress pathways.

Key words: Ricin toxin, Pyroptosis, Oxidative stress, Circular RNA, Competing endogenous RNA