Preliminary Study On The Relationship Between The Regulatory Effect Of EZH2 Gene Ferroptosis And The Radiosensitivity Of KDM6A-deficient ESCC Cells

Backgound EZH2(enhancer of zeste homolog 2)is a gene with histone methyltransferase activity that may trimethylate lysine at the 27 th position of histone H3(H3K27me3),hence limiting the transcription of target genes.KDM6 A is an essential EZH2 antagonist gene.Under typical conditions,the two functions are in dynamic equilibrium.When KDM6 A is lacking or EZH2 is overexpressed,this equilibrium is disrupted,resulting in the development of different cancers.According to existing studies,EZH2 has become a therapeutic target for a range of malignancies.Although KDM6 A and EZH2 have been researched to varying degrees in several cancers,esophageal squamous cell carcinoma research is still lacking(ESCC).In this work,EZH2 and KDM6 A were paired with radiation to investigate the mutation of KDM6 A in esophageal squamous cell carcinoma(ESCC)and the effect of targeted EZH2 on the radiosensitivity of KDM6A-deficient ESCC and its mechanism.Objective To investigate the relationship between the regulation of EZH2 on ferroptosis and the radiosensitivity of KDM6A-deficient esophageal squamous cell carcinoma.Methods Using Western blot,the mutation of KDM6 A in esophageal squamous cell carcinoma cells was detected.We chose the KDM6A-deficient cell line KYSE180 and the wild-type cell line TE1.Using lentiviral interference technique,EZH2 stable silencing lines were constructed,or the EZH2 inhibitor Tazemetostat(EPZ-6438)was utilized to block EZH2.With irradiation,EPZ-6438 IC50 and G2/M cell cycle arrest were observed in ESCC cells.Using a plate colony formation test,the number of cell colonies in each group was determined.Using the linear quadratic model(L-Q model)to match the cell survival rate,the radiosensitivity enhancement ratio(SER)was obtained.The effect of silencing or inhibiting EZH2 on the radiosensitivity of KDM6A-deficient ESCC cells was then measured.The expression of γ-H2 AX and53BP1 in each set of cells was detected by immunofluorescence,and the DNA damage in each group was compared.In KDM6A-deficient ESCC cells,the effect of EZH2 interference or inhibition on radiation-induced DNA damage was investigated.Using differential analysis and enrichment analysis,regulatory pathways of the radiosensitizing impact of EZH2 silencing on KDM6A-deficient ESCC cells were screened.The level of PTGS2 in each group was measured,and the effect of suppressing or interfering with EZH2 on PTGS2 in KDM6A-deficient ESCC cells was determined.Result Western blotting revealed that KDM6 A mutations occur often in ESCC.The IC50 of EPZ-6438 revealed that KDM6 A wild-type ESCC cell lines were more resistant to EPZ-6438 than ESCC cell lines with a KDM6 A deficiency.According to the outcomes of the G2/M cell cycle arrest test and the plate cloning experiment,interference with EZH2 or suppression of EZH2 considerably improved the radiosensitivity of KDM6A-deficient KYSE180 cells but had no influence on the radiosensitivity of TE1 cells.Immunofluorescence labeling for γ-H2 AX and 53BP1 revealed that interference or suppression of EZH2 greatly exacerbated the radiation-induced DNA damage in KYSE180 cells relative to TE1 cells.By activating the ferroptosis pathway,EZH2 knockdown improved the radiosensitivity of KDM6A-deficient ESCC KYSE180 cells using differential and enrichment analyses.Detecting PTGS2 in cells revealed that inhibition or interference of EZH2 raised the amount of PTGS2 in KDM6A-deficient cell lines but not in wild-type cells.Conclusion Mutations of KDM6 A are prevalent in esophageal squamous cell carcinoma tissues and cells.EZH2 silencing dramatically increases the radiosensitivity of KDM6 A mutant ESCC cells to radiation.Interference with EZH2 can increase the activation of the radiation-induced ferroptosis pathway,indicating that inhibiting EZH2 increases the radiosensitivity of KDM6A-deficient ESCC cells by enhancing the radiationinduced ferroptosis effect.