Study On The Antitumor Mechanism Of Histone Methyltransferase Inhibitor DCG066 To Lymphoma

Objectve:Diffuse large B-cell lymphoma(DLBCL)is a highly malignant lymphoma,and the conventional R-CHOP treatment regimen is ineffective and cannot completely remove drug-resistant tumor cells.Therefore,it is urgent to start from the pathogenesis of symptomatic treatment and explore new rituximab combination regimens to increase the therapeutic effect on relapsed and refractory lymphoma.Mutations or rearrangements of methyltransferase G9 a,tumor suppressor gene p53 and anti-apoptosis gene Bcl-2 can be detected in DLBCL patients,which play an important role in the development of DLBCL.Therefore,this study intends to start from the pathogenesis of DLBCL,explore the effective killing concentration of histone methyltransferase inhibitor DCG066 on DLBCL cell line TMD-8,and then study its effect on DLBCL in cell proliferation,cell cycle,apoptosis and ferroptosis and analyze the mechanism of action and key functional molecules.Methods:(1)The effective killing concentration of DCG066 on TMD-8 cells was explored by CCK-8 experiment.(2)The distribution of TMD-8 live and dead cells after DCG066 treatment was analyzed by Calcein-AM/PI live/dead cell double staining kit combined with fluorescence assay.(3)The apoptosis induction of TMD-8 cells by DCG066 was detected by Annexin-V-FITC/PI double staining apoptosis kit combined with flow cytometry.(4)The inhibition of TMD-8 cell cycle by DCG066 was detected by cell cycle detection kit combined with flow cytometry.(5)The induction of reactive oxygen species(ROS)in TMD-8 cells by DCG066 was detected by reactive oxygen species detection kit combined with flow cytometry.(6)RNA was extracted and sent to transcriptome sequencing to analyze the differential expression of apoptosis and ferroptosis-related genes.(7)Total protein was extracted,and Western Blot experiments were performed to verify the changes in the expression levels of key molecules of apoptosis and ferroptosis.(8)SPSS 22.0 software was used for data analysis,the experimental data were expressed as x ±s,and the t test was used for comparison between the two groups,and P<0.05 was considered statistically significant.Results:(1)After DCG066 treatment,the activity of TMD-8 cells was significantly decreased,and the proportion of dead cells was significantly increased in a dose-dependent manner.(2)After DCG066 treatment,the proportion of cells in G0/G1 phase increased,the proportion of cells in G2/M phase decreased,and the expressions of cyclins Cyclin A2 and Cyclin E1 were down-regulated in TMD-8 cells.(3)After DCG066 treatment,the apoptosis rate of TMD-8 cells increased,the expressions of apoptosis inhibitory genes Bcl-2,BCL2A1 and IL3 RA were down-regulated,and the expressions of pro-apoptotic genes p53,Bax,Cleave-Caspase,CTSO,ERN1 and DDIT3 were up-regulated.(4)After DCG066 treatment,the level of ROS in TMD-8 cells increased,the expression of ferroptosis-related genes NOX2,SAT2,HMOX1 was up-regulated,and the expression of SLC7A11 was down-regulated.Fer-1,an inhibitor of ferroptosis,could reverse the increase of ROS level.Conclusion:(1)Histone methyltransferase methyltransferase DCG066 can effectively kill DLBCL cells in a dose-dependent manner.(2)DCG066 inhibits the proliferation of DLBCL cells by blocking the cell cycle in G2/M phase by downregulating cyclin.(3)DCG066 can induce apoptosis in DLBCL cells by down-regulating the expression of apoptosis-inhibiting genes and up-regulating the expression of pro-apoptotic genes.(4)DCG066 can accumulate ROS in DLBCL cells by up-regulating the expression of ferroptosis-related genes,and finally lead to the occurrence of ferroptosis.In conclusion,the histone methyltransferase inhibitor DCG066 can kill diffuse large B-cell lymphoma by inhibiting cell proliferation,inducing apoptosis and ferroptosis.