Effect Of NSD2 Silencing On Gene Expression Profile And Biological Behavior Of Triple Negative Breast Cancer

Objective: In this study,the expression of NSD2 was silenced in triple negative breast cancer(TNBC)cell line MDA-MB-231,and the effects of NSD2 on the biological behavior of TNBC cells and drug sensitivity were observed by combining gene chip,bioinformatics methods and in vitro experiments,so as to explore the possible role and mechanism of NSD2 in TNBC.Methods: Cell lines with silenced NSD2 expression were constructed,and gene chip technology was used to detect the changes in gene expression profile of MDA-MB-231 after silenced NSD2 expression,and the genes with significant differences were screened out.KEGG,David,String and Cytoscape were used to analyze the differential genes and screen the relevant Hub genes.Kaplan-Meier Plotter was used to analyze the prognosis of the Hub genes.CCK-8 cell proliferation assay,Hoechst(33258)cell apoptosis staining assay,flow cytometry and Western Blot immunoblotting assay were used to detect the changes in biological behavior and death receptor-related protein expression of TNBC cells after knockdown of NSD2.Results: The MDA-MB-231 cell line with silenced NSD2 expression was successfully constructed.After silenced NSD2 expression,the expression profile of its TNBC cells was significantly changed.A total of 483 differential genes were found,including 324 up-regulated genes and 159 down-regulated genes.KEGG pathway enrichment analysis of differential genes showed that NSD2-related differential genes involved 30 signaling pathways,such as cytokine-cytokine receptor interaction,necroptosis,and TNF signaling pathway.GO functional enrichment analysis showed that the upregulated genes were enriched in 79 BP,14 MF and 29 CC,while the down-regulated genes were enriched in 166 BP,36 MF and 56 CC(P<0.05),in which differential gene enrichment pointed to multiple tumor GO processes.Hub genes were screened by Cytohubba in Cytoscape for PPI network of significantly different genes,and a total of 25 Hub genes were screened out.After Kaplan-Meier Potter prognostic analysis,16 NSD2-related differential genes,including CCL5,CXCL10,CXCL11,IFIT1,IFIT2,IFIT3,OAS1,OAS2,IRF1,ISG20,IFIH1,RSAD2,THBS1,DDX58,MX1,HERC6,were found to be significantly correlated with the prognosis of TNBC(P<0.05).The results of CCK-8 assay showed that PTX could inhibit cell proliferation in a dose-dependent and time-dependent manner.After silenced NSD2 expression,cell survival in the sh NSD2-1 group and the sh NSD2-2 group decreased compared to the sh NC group.The results of Hoechst(33258)staining and flow cytometry showed that the apoptosis rate was increased in the PTX group compared with the untreated group.In the untreated group,the apoptosis rate of sh NC group,sh NSD2-1 group and sh NSD2-2 group had no difference.In the PTX treated group,compared with the sh NC group,the apoptosis rate of sh NSD2-1 and sh NSD2-2 groups increased,while the number of cells decreased.Western Blot results showed that the expression of cell death signaling pathway related proteins in MDA-MB-231 cells increased after silenced NSD2.In the untreated group,compared with the sh NC,the expression of TNFAIP3,DR5,Fas protein in the sh NSD2-1 group was significantly increased(P<0.05),the expression of CASP-1 was slightly increased,the expression of PARP1 was slightly decreased(P<0.05),and the expression of TRAIL was slightly decreased,but Cleaved PARP1 was not significantly changed.The protein expression levels in the sh NSD2-2group were all increased,and the difference was statistically significant(P<0.05).In the PTX group,compared with the untreated group,the protein expression levels were significantly increased(P<0.05).Compared with sh NC+PTX group,the expression of TNFAIP3,DR5,CASP-1,Fas protein in sh NSD2-1+PTX group was significantly increased(P<0.05),the expression of Cleaved PARP1,TRAIL was increased,and the expression of PARP1 was slightly decreased.All the protein expression levels in sh NSD2-2+PTX group were increased(P<0.05).Prognostic analysis of the above proteins showed that,except for PARP1(P<0.05;HR>1),the high expression of other 5proteins all suggested a good prognosis(P<0.05;HR<1).Conclusions:1.NSD2 silencing can significantly change the gene expression profile of TNBC cells,and NSD2-related differential genes are involved in multiple tumor-related signaling pathways.2.Sixty NSD2-related differential genes,including CCL5,CXCL10,CXCL11,IFIT1,IFIT2,IFIT3,OAS1,OAS2,IRF1,ISG20,IFIH1,RSAD2,THBS1,DDX58,MX1 and HERC6,were identified as potential biomarkers for predicting the prognosis of TNBC.3.NSD2 silencing can inhibit the proliferation ability of TNBC cells and enhance their drug sensitivity to PTX.NSD2 may mediate PTX drug resistance in patients with TNBC,and NSD2 silencing combined with PTX treatment may change the prognosis of patients.4.The synergistic effect between NSD2 silencing and PTX can induce the death of TNBC cells.NSD2 may enhance the proliferation ability of TNBC cells and inhibit their death by inhibiting apoptosis pathway mediated by death receptor and classical pyroptosis pathway mediated by CASP-1.5.TRAIL/DR5,Fas,CASP-1 and TNFAIPP3 may be closely related to breast cancer,and their increased expression is positively correlated with the prognosis of patients,which may be used as molecular targets for predicting the prognosis of breast cancer.