| Objective:The purpose of this study is to examine the expression levels of EZH2(enhancer of zeste homolog2)and SETD2(SET domain containing2)in nasopharyngeal carcinoma(NPC)tissues and NPC cell lines.Moreover,the study aims to investigate the impact of SETD2 on the migration and invasion capability of NPC cells.Additionally,this research intends to conduct a preliminary analysis of the protein-level interaction between SETD2 and EZH2,which may affect the malignant biological behavior of NPC cells.Methods:This study utilized the m RNA expression database of Head and Neck Squamous Cell Carcinoma(HNSCC)from The Cancer Genome Atlas(TCGA)database for the analysis of differential genes using the R language package edge R.To further investigate the m RNA and protein expression levels of SETD2 and EZH2 genes,real-time quantitative reverse transcription polymerase chain reaction(RT-q PCR)and Western blot(WB)techniques were employed for clinical NPC tissues and nasopharyngeal chronic inflammation tissues.High-invasive human NPC cell lines5-8F and CNE1 were also used for cell experiments to detect the m RNA and protein expression levels of SETD2 and EZH2 in NP69,5-8F,and CNE1,utilizing RT-q PCR and WB techniques.Stable overexpression and knockdown SETD2 nasopharyngeal carcinoma cell lines were constructed via CRISPR-Cas9 SAM genome editing technology and lentivirus infection technology,and the m RNA and protein expression levels of SETD2 and EZH2 were detected through RT-q PCR and WB techniques.The direct interaction between SETD2 and EZH2 was assessed using co-immunoprecipitation(COIP)-WB technique.Additionally,scratch test,Transwell migration,and invasion experiments were carried out to determine the migration and invasion abilities of overexpressed and knockdown SETD2 nasopharyngeal carcinoma cell lines.Results:(1)Based on the analysis of the TCGA database,it was found that the expression of SETD2 was significantly lower in HNSC than in the normal group(P<0.05).In contrast,the expression of EZH2 was significantly higher in HNSC than in the normal group(P<0.05);(2)The results of the RT-q PCR assay indicated that the relative m RNA expression of SETD2 was lower in NPC clinical tissues(0.52±0.21)compared to chronic inflammation tissues(1.12±0.32).Conversely,the relative m RNA expression of EZH2 was higher in NPC clinical tissues(2.35±0.41)than in chronic inflammation tissues(1.12±0.24).Additionally,in 5-8F and CNE1 cells,the relative m RNA expression of SETD2 was significantly lower than that in NP69 cells,whereas the relative m RNA expression of EZH2 was significantly higher than that in NP69 cells(P<0.01).(3)According to the results of WB analysis,the protein expression level of SETD2 was observed to be lower in clinical nasopharyngeal carcinoma(NPC)tissues in comparison to chronic inflammation tissues.In contrast,the protein expression level of EZH2 was found to be higher in NPC clinical tissues when compared to chronic inflammation tissues(P<0.01).(4)In addition,when comparing 5-8F and CNE1 cells to NP69 cells,the ralative m RNA expression and the protein expression level of SETD2 was found to be lower while that of EZH2 was higher with statistically significant differences(P<0.01).(5)Validation was performed using RT-q PCR and WB,following overexpression of SETD2 in NPC,which indicated a significant increase in the protein expression level of SETD2 and a noteworthy decrease in the protein expression level of EZH2(P<0.01).These changes were observed to have statistically significant differences.However,no significant difference was observed in the relative m RNA expression levels of SETD2 and EZH2.(6)Validation was performed by RT-q PCR and WB following the knockdown of SETD2 in NPC.The results showed a substantial decrease in the protein expression level of SETD2 and a noteworthy increase in the protein expression level of EZH2(P<0.001).These alterations were found to have statistically significant differences.However,there was no significant difference observed in the relative m RNA expression levels of SETD2 and EZH2.(7)The COIP-WB assay findings indicate that the interaction between SETD2 protein and EZH2 protein is observable in OE-SETD2-CNE1 cells.(8)The results of scratch assays demonstrated that,at the 24-hour time point,the migration rate of NPC cells overexpressing SETD2 was lower than that of the NC group(0.46±0.02 VS 0.17±0.02,P<0.05),whereas the migration rate of NPC cells with SETD2 knockdown was higher than that of the NC group(0.34±0.02 VS0.66±0.02,P<0.05).Furthermore,the Transwell invasion assay results indicated that the number of invaded cells within 24 hours was lower in SETD2 overexpression NPC cells than in the NC group(126±7 VS 84±10,P<0.05),whereas the number of invaded cells within 24 hours was higher in SETD2 knockdown NPC cells than in the NC group(123±10 VS 143±11,P<0.05).These observed differences in invasion rates between the groups were found to be statistically significant(P<0.05).Conclusions:(1)NPC tissues and cell lines exhibit a low SETD2 expression and a high EZH2expression;(2)The protein-protein interaction between SETD2 and EZH2 has been shown to restrain the migration and invasion capabilities of NPC cells. |
