Regulation Of RNA Methylation And Secretion Of Inflammatory Factors By 5-Azacytidine In SARS-CoV-2 Virus

Objective: This study used epigenetic therapy to investigate the regulation of SARS-Co V-2 viral RNA methylation by hypercytokinaemia through molecular biology,cell biology and genomic approaches to inhibit viral replication and alleviate immune factor secretion,thereby providing new potential therapeutic options for COVID-19.Methods: 1.BHK21-h ACE2 cells were infected with packaged pseudovirus VSV-SARSCo V-2-Sdel18.Three groups of 5-azacytidine,chloroquine and 5-azacytidine combined with chloroquine were set up to detect the inhibitory effect on the viruses respectively.2.The viral RNA was bisulfite converted and sequenced on the MGISEQ-2000 platform,5m C level of RNA was counted,and a comparative study was performed with chloroquine.3 The S CAR-T assay was used for cytokine ELISA to observe the effects of concentration gradients of 5-azacytidine on hypercytokinaemia.4 Transcriptome sequencing of cell RNA was carried out to performing the differential genes expression analysis between the 5-azacytidine treatment group and the control group,and followed by the enrichment analysis based on these differential genes.Results: 1.Cells treated with 5-azacytidine showed significantly reduced fluorescence intensity.No significant difference was observed between the combination of 5-azacytidine and chloroquine compared to the single drug.2.Total RNA 5-methylcytosine modification was decreased with 5-azacytidine treatment,and 5-methylcytosine levels were significantly reduced by 23.47% in 5-azacytidine-treated samples.3.The secretion of inflammatory factors IL-6 and TNF-α was inhibited after 5-azacytidine treatment,and decreased significantly with the increase of drug concentration.4.5-azacytidine treatment led to changes in gene expression of cells,which were found to be mainly enriched in infection and immune related pathways by KEGG Pathway enrichment analysis of differentially expressed genes.Conclusion: In this study,we found 5-azacytidine inhibited VSV-SARS-Co V-2-Sdel18 and effectively suppressed the methylation of total RNA,which is similar to chloroquine,but the cytotoxicity is higher than chloroquine at higher concentrations.5-azacytidine is mainly incorporated into the RNA of the virus and prevents viral replication by interfering with the modification of 5-methylcytosine.For the explosive increase of inflammatory factors caused by the new coronavirus,5-azacytidine has a certain inhibitory effect.