Anti-Tumor Effect And Mechanism Of Chidamide In Diffuse Large B-Cell Lymphoma

ObjectiveThe purpose of this study was to investigate the effects of chidamide on proliferation and apoptosis of diffuse large B-cell lymphoma U2932(ABC subtype)and SUDHL-4(GCB subtype)cells,and to explore the mechanism of chidamide acting on ABC and GCB subtypes.The therapeutic effect and mechanism of chidamide on ABC and GCB subtypes were compared.Method1.CCK8 was used to detect the effect of chidamide on the viability of U2932 and SUDHL-4cells.2.Flow cytometry was used to detect the effect of chidamide on apoptosis of U2932 and SUDHL 4 cells.3.U2932 cells and SUDHL-4 cells were treated with 4 umol /l chidamide,The samples of U2932 cells,SUDHL-4 cell chidamide-treated group and control group were collected and three biological replicates were performed.The collected 12 samples were extracted for m RNA sequencing.The m RNA sequencing results of U2932 cells,SUDHL-4 cell chidamide-treated group and control group were analyzed by difference analysis,GO,KEGG and GSEA enrichment analysis,to explore the mechanism of chidamide acting on DLBCL.4.Cytoscape was used to analyze the protein interactions of different genes in U2932 cells,SUDHL-4 cell chidamide-treated group and control group,to find out the key subnetworks and key genes,and key genes are used to predict the effect of chidamide.5.The common differential genes of U2932 and SUDHL-4 cells were identified,and the correlation analysis of the common differential genes was conducted.Result1.The 24 h proliferation inhibition rate of U2932 cells was decreased from(74.30±5.81)% to(37.86±5.86)% after treatment with different concentrations of chidamide(30 umol/ L,15 umol/ L,7.5 umol/ L,3.75 umol/ L,1.875umol/L).The difference was statistically significant(P <0.001).After 48 h treatment,the proliferation inhibition of U2932 cells decreased from(91.50±1.11)% to(37.16±2.43)%,the difference was statistically significant(P <0.001).The IC50 values of U2932 cells treated with chidamide at 24 h and 48 h were(3.74±0.03)umol/ L and(2.55±0.17)umol/ L,and the difference was statistically significant(P <0.01).2.The proliferation inhibition rate of SUDHL-4 cells decreased from(76.35±1.07)% to(30.75±1.08)% at 24 h after treatment with different concentrations of chidamide(30 umol/ L,15 umol/ L,7.5 umol/ L,3.75 umol/ L,1.875umol/L).The difference was statistically significant(P<0.001).The proliferation inhibition rate of SUDHL-4 cells after 48 h treatment decreased from(90.32±1.25)% to(32.05±2.76)%,and the difference was statistically significant(P <0.001).The IC50 values of SUDHL-4 cells treated with chidamide at 24 h and 48 h were(5.44±0.11)umol/L and(3.87±0.30)umol/L,the difference was statistically significant(P <0.001).3.There were significant differences in the IC50 of chidamide at 24 hours(P < 0.001)and at 48 hours(P < 0.01)between U2932 cells and SUDHL-4cells.4.The 48 h apoptosis rate of U2932 cells treated with different concentrations of chidamide(0umol/l,1.1 umol/l,3.3 umol/l,10 umol/l,30umol/l)increased from(6.31±0.81)% to(42.60± 2.22)%,the difference was statistically significant(P <0.05).The 48 h apoptosis rate of SUDHL-4 cells increased from(8.27±0.76)% to(65.13±9.24)%,the difference was statistically significant(P <0.05).5.Compared with the control group,1255 up-regulated genes and 6down-regulated genes were found in chidamide treated U2932 cells.GSEA enrichment was significantly enriched in PI3K-Akt signaling pathway when the tumor-related pathway gene set was used as reference.Compared with the control group,there were 1686 up-regulated genes and 656 down-regulated genes in chidamide SUDHL-4 cells,and the GSEA enrichment was significant in PI3K-Akt signaling pathway when the tumor-related pathway gene set was used as a reference.6.In U2932 cells,10 key genes of protein interaction related to chidamide treatment,ALB,FN1,SRC,CXCL8,FGF2,PTK2,PECAM1,BDNF,JUN and CXCL10,were found.In SUDHL-4 cells,10 key genes of protein interaction related to chidamide treatment,TP53,FN1,BRCA1,CCND1,CDC6,CDK2,MCM2,PCNA,MCM7 and JUN,were identified.The high expression of key node genes FN1,PTK2 and JUN of U2932 cells may indicate that DLBCL is resistant to chidamide.The high expression of key node genes FN1,CCND1 and JUN of SUDHL-4 cells may indicate that DLBCL is resistant to chidamide,and the high expression of BRCA1,MCM2 and MCM7 may indicate that DLBCL is sensitive to chidamide.7.There was no significant difference in the expression of BCL2 and MYC genes in U2932 cells between the chidamide treatment group and the control group(BCL2 P >0.05,MYC P >0.05).There was no significant difference in the expression of BCL2 and MYC genes in SUDHL-4 cells between the chidamide treatment group and the control group(BCL2 P >0.05,MYC P >0.05).8.By comparing the differential genes of U2932 and SUDHL-4 cells treated with chidamide,402 same differential genes were found,but correlation analysis showed that log2 FC correlation of these genes was weak.Conclusion1.chidamide can inhibit the proliferation of ABC subtype(U2932)and GCB subtype(SUDHL-4)DLBCL cells in a time and concentration dependent manner.The inhibitory effect of chidamide on the proliferation of ABC subtype(U2932)was better than that of GCB subtype(SUDHL-4).2.chidamide can promote apoptosis of ABC subtype(U2932)and GCB subtype(SUDHL-4)DLBCL in a time and concentration dependent manner.3.chidamide may have an anti-tumor effect on DLBCL cells by affecting the PI3K-Akt pathway.4.The high expression of key node genes FN1,PTK2 and JUN of ABC subtype may indicate that DLBCL is resistant to chidamide.The high expression of the key node genes FN1,CCND1 and JUN of the GCB subtype may indicate that DLBCL is resistant to chidamide,and the high expression of BRCA1,MCM2 and MCM7 may indicate that DLBCL is sensitive to Chidamide.5.The expression levels of BCL2 and MYC in ABC and GCB subtypes did not change significantly after treatment with Chidamide.