Role And Mechanism Of DNMT1 In Regulating Pancreatic Cancer Cells Ferroptosis

Objective:Ferroptosis is a new form of cell death,and the factors effecting ferroptosis sensitivity varies among tumor cells.In this study,we investigated the role of DNA Methyltransferase 1（DNMT1）in regulating ferroptosis susceptibility of pancreatic cancer cells in order to provide new strategies for molecular typing and targeted therapy of pancreatic cancer.Methods:（1）Analysis of DNMT1 expression levels,clinical significance and its correlation with ferroptosis in pancreatic cancer tissues and cell lines using bioinformatics methods and Western blot.（2）Validation of the efficiency of DNMT1 knockdown of pancreatic cancer cell lines using Western blot and q RT-PCR.（3）Detection of cell viability,clonogenic ability,intracellular reduced glutathione level,lipid reactive oxygen species levels,malondialdehyde levels and free Fe²⁺levels in DNMT1 knockdown cells using CCK8 assay,clonogenic formation assay,glutathione assay kit,C11-BODIPY dye,malondialdehyde assay kit and iron assay kit,respectively.（4）RNA sequencing technology（RNAseq）and whole genome simplified methylation sequencing（RRBS）were used to screen DNMT1 downstream regulatory molecules;（5）Western blot and q RT-PCR were used for validating DNMT1 downstreaming regulators.Results:（1）CCLE database analysis showed that DNMT1 was widely expressed in pancreatic cancer cells.TCGA database analysis showed that DNMT1 expression in tumor tissues was significantly higher than that in normal tissues,and DNMT1 expression was negatively correlated with patient survival,positively correlated with mesenchymal molecules,and positively correlated with ferroptosis-positive molecules;（2）Western blot and q RT-PCR results suggested the successful construction of DNMT1knockdown pancreatic cancer cell lines;（3）The cell viability and clonogenic capacity of DNMT1 knockdown pancreatic cancer cells were higher than those of the control group,with no significant changes in intracellular glutathione levels and significantly lower levels of lipid reactive oxygen species,malondialdehyde and Fe²⁺level than those of the control group in erastin or RSL3 induced ferroptosis.（4）DNMT1 inhibitor zebraline also significantly increased cell viability in ferroptosis.（5）The results of RNAseq suggested that knockdown of DNMT1 resulted in the differential expression of a total of 1405 genes,of which 1314 were up-regulated and91 were down-regulated.RRBS sequencing results showed that knockdown of DNMT1resulted in almost genome-wide down-regulation of methylation.（6）The results of RRBS combined with RNAseq analysis suggested that there were1024 genes in the intersection of differential gene intersections where demethylation and elevated gene expression occurred after knockdown of DNMT1,of which the top100 genes intersected with only one ferroptosis-associated gene,FTH1..（7）Western blot and q RT-PCR showed that the FTH1 protein and RNA levels were significantly increased after knockdown of DNMT1.Conclusions:DNMT1 plays a key role in regulating ferroptosis sensitivity in pancreatic cancer cells.DNMT1 regulates the transcriptional expression level of FTH1 by maintaining the level of methylation in its promoter region.Inhibition of DNMT1 can downregulate the methylation level of the FTH1 promoter region and upregulate the expression level of FTH1,causing a decrease in intracellular Fe²⁺levels and ultimately leading to cell ferroptosis resistance.