| Nasopharyngeal carcinoma(NPC)is a malignant tumor that originates from the mucosa of the nasopharynx,with the majority of cases occurring in the Guangdong and Guangxi provinces of China and Southeast Asia.It is a highly malignant cancer with a tendency to metastasize,posing a serious threat to the physical and mental health of patients.Numerous studies have shown that the expression of various oncogenes induced by Epstein-Barr virus(EBV)infection is the main cause of NPC.In recent years,it has been found that the expression of GSG2(Haspin)is upregulated in many tumors,and its expression level is correlated with tumor occurrence,development,and prognosis.However,research on the role of GSG2 in NPC has not been reported.Although recent literature reports high expression of GSG2 in gallbladder cancer,prostate cancer,and breast cancer,where it regulates tumor cell proliferation,and reports that GSG2 promotes the development of bladder cancer by targeting KIF15,its role in NPC has never been studied,and there is no research on the correlation between GSG2 and classical tumor signaling pathways.Therefore,this study aims to first confirm the expression of GSG2 in NPC and its clinical relevance,explore the relationship between the expression of GSG2 and biological phenotypes in NPC cells,and attempt to find the mechanism by which GSG2 promotes the malignant biological behavior of NPC cells through classical tumor signaling pathways,in order to find new potential targets for the treatment of NPC.Objective:The aim of this study is to first clarify the expression characteristics of GSG2 in nasopharyngeal carcinoma tissue and its clinical significance.Then,stable knockdown and overexpression nasopharyngeal carcinoma cell lines will be established,and classic biological methods will be used in vitro and in vivo to study the regulatory effects of GSG2 through the Wnt/β-catenin signaling pathway on the proliferation,migration,and invasion ability of nasopharyngeal carcinoma cells.Finally,the effectiveness of CHR-6494,a GSG2(Haspin)protein kinase inhibitor,as a target for nasopharyngeal carcinoma cell therapy will be studied.It is hoped that this study will provide new molecular mechanisms for the regulation of GSG2 in nasopharyngeal carcinoma,identify relevant molecular markers,and lay a theoretical foundation for further research on the diagnosis,treatment,and prognosis of nasopharyngeal carcinoma.Methods:1.The expression level of GSG2 in nasopharyngeal carcinoma was analyzed using GEO database chip analysis,and the expression level of GSG2 was determined by immunohistochemistry in 101 cases of nasopharyngeal carcinoma and 30 cases of adjacent tissues in tissue chips.Statistical software was used to correlate the expression level of GSG2 with clinical data of patients.2.The cell lines 5-8F,C666-1,and HONE-1 were cultured,and cell lines with significant expression of GSG2 were selected.Three RNA interference target sequences were designed for endogenous screening,and the plasmid with the highest knockdown efficiency was selected,packaged into a lentivirus,and infected into nasopharyngeal carcinoma cell lines to establish stable transfection cell lines with specific knockdown of GSG2 and their control cell lines.q PCR and Western blot methods were used to detect the knockdown efficiency of GSG2.3.MTT assay,cell apoptosis assay,and cell colony formation assay were used to observe the effect of GSG2 knockdown on the in vitro proliferation ability of nasopharyngeal carcinoma cells using sh GSG2 and empty vector control groups.4.Nude mouse subcutaneous tumor experiments were conducted by transfecting sh GSG2 and empty vector control C666-1 cells.The weight,length,and width of the tumors were recorded at different time points,and the tumor volume was calculated.The fluorescence intensity of the tumors was measured using a small animal live imaging instrument,and all mice were euthanized.The tumors were removed,photographed,weighed,and recorded,and immunohistochemistry was used to detect the expression of Ki67 in the tumor tissue.The effect of GSG2 knockdown on the in vivo proliferation ability of nasopharyngeal carcinoma cells was observed.5.Transwell and scratch assay were used to observe the effect of GSG2 knockdown on the in vitro invasion and migration ability of nasopharyngeal carcinoma cells using sh GSG2 and empty vector control groups.6.The coexpedia website was used to search for GSG2 co-expressed genes.KEGG analysis was then performed to identify the relevant signaling pathways with high correlation.Finally,the IPA software was used to establish a network of GSG2,its downstream genes,and the aforementioned pathways.7.GSG2-related signaling pathways were selected based on bioinformatics analysis,and the signaling pathways closely related to the occurrence and development of nasopharyngeal carcinoma were identified.Western blot was used to further verify the changes in protein expression of key target genes in the signaling pathway after GSG2 knockdown.8.A lentivirus overexpressing GSG2 was constructed and transfected into C666-1cells to obtain a stable transfected nasopharyngeal carcinoma cell line.MTT,apoptosis assay,scratch assay,and Transwell assay were used to detect the effects of GSG2 overexpression and addition of the Wnt/β-catenin signaling pathway inhibitor(C59)on the proliferation,migration,and invasion of nasopharyngeal carcinoma cells.The results confirmed that GSG2 promotes the malignant biological behavior of nasopharyngeal carcinoma cells through the Wnt/β-catenin signaling pathway.9.Transfected C666-1 cells with GSG2 or empty vector were used for subcutaneous tumorigenicity assay in nude mice.The body weight,tumor length and width were recorded at different time points,and tumor volume was calculated.The fluorescence intensity of tumors was measured by a small animal in vivo imaging system.All mice were sacrificed and the tumors were removed for photography,weighing and immunohistochemical analysis of Ki67 expression to observe the effect of GSG2 overexpression and the addition of Wnt/β-catenin signaling pathway inhibitor(C59)on the in vivo proliferation ability of nasopharyngeal carcinoma cells.10.GSG2 and empty vector control cells were used to verify the changes in expression of key target genes in the Wnt/β-catenin signaling pathway after GSG2 overexpression and addition of GSG2(Haspin)protein kinase inhibitor CHR-6494 by Western blot.MTT and cell apoptosis assays were used to observe the effect of GSG2 overexpression and the addition of CHR-6494 on the in vitro proliferation ability of nasopharyngeal carcinoma cells.Results:1.GSG2 expression was found to be higher in advanced nasopharyngeal carcinoma(NPC)compared to early stage NPC(P<0.05);GSG2 expression was significantly higher in NPC tissues compared to adjacent noncancerous tissues(P<0.001);and GSG2 expression was positively correlated with gender,with higher expression observed in female patients(P<0.05).These findings are consistent with the results of comparing GSG2 expression profiles in NPC and adjacent noncancerous tissues in the GEO database.2.Using stable GSG2 knockdown cell lines,MTT,colony formation,and apoptosis assays revealed that GSG2 knockdown significantly inhibited in vitro proliferation of nasopharyngeal carcinoma cells(C666-1 group P<0.001,HONE-1 group P<0.001).GSG2 knockdown also successfully inhibited in vivo tumor growth of C666-1 cells in a nude mouse model,as evidenced by reduced fluorescence intensity,size,weight,and Ki67 expression in tumor tissues(P<0.05).3.Transwell and scratch experiments were used to detect that GSG2 knockdown had a significant inhibitory effect on cell migration and invasion abilities(C666-1 group P<0.001,HONE-1 group P<0.001).4.IPA network analysis established a molecular and signaling pathway network associated with GSG2,and found that the Wnt/β-catenin signaling pathway may be closely related to the regulation of nasopharyngeal carcinoma development by GSG2.Western blot analysis of key target gene proteins in the Wnt/β-catenin signaling pathway in GSG2 overexpressing and knockdown stable transfection cell lines showed that GSG2 protein expression was upregulated,and β-Catenin,p-β-Catenin,Smad4,LEF1,and Wnt1 protein expression also increased(P < 0.05);GSG2 protein expression was downregulated,and β-Catenin,p-β-Catenin,Smad4,LEF1,and Wnt1 protein expression also decreased(P<0.05).5.The GSG2 overexpression stable transfection cell line was constructed,and MTT and apoptosis experiments were used to detect that GSG2 overexpression enhanced cell proliferation ability(P < 0.001).Addition of C59 reduced the difference in cell proliferation ability between the GSG2 overexpression group and its control group of in vitro nasopharyngeal carcinoma cells(P<0.001).Successful subcutaneous tumorigenesis of the GSG2 overexpression cell line in nude mice was achieved,and detection of fluorescence intensity,size,weight,and Ki67 expression in tumor tissues showed that GSG2 overexpression also enhanced the proliferation ability of nasopharyngeal carcinoma cells in vivo,and addition of C59 reduced the difference in proliferation ability between the GSG2 overexpression group and its control group(P<0.05).6.Through Transwell and scratch assays,it was found that overexpression of GSG2 gene significantly enhanced the migration and invasion ability of nasopharyngeal carcinoma cells(P < 0.001).After adding C59,the difference in migration and invasion ability between the GSG2 overexpression group and its control group in vitro was reduced(P < 0.001).7.The stable transfection cell line overexpressing GSG2 was examined using MTT and apoptosis assays,and it was found that the proliferation ability of C666-1 cells overexpressing GSG2 was enhanced(P < 0.001).After adding CHR-6494,the difference in proliferation ability between the GSG2 overexpression group and the control group of nasopharyngeal carcinoma cells was reduced(P < 0.001).By Western blotting,key target gene proteins of the Wnt/β-catenin signaling pathway were detected,and it was found that the addition of CHR-6494 could reverse the increase in protein expression of p-STAT1,p-ERK,p-P38,LEF1,Wnt1,SMAD4,and p-β-catenin caused by GSG2overexpression(P < 0.001).Conclusion:GSG2 is highly expressed in nasopharyngeal carcinoma tissue,and its expression increases with the later stages of the disease.The expression of GSG2 is higher in female patients than in male patients,and there is a correlation with patient gender.In terms of nasopharyngeal carcinoma cells,the Wnt/β-catenin pathway may be a direct target of GSG2 regulation,and GSG2 can promote nasopharyngeal carcinoma cell proliferation,migration,and invasion through the Wnt/β-catenin pathway.The protein kinase inhibitor CHR-6494 also has the same effect of reducing nasopharyngeal carcinoma cell proliferation by knocking down the GSG2 gene,and therefore may have great potential as a molecular targeted drug for nasopharyngeal carcinoma.Our results confirm that GSG2 is a molecular marker closely related to nasopharyngeal carcinoma,and lay a theoretical foundation for the in-depth study of molecular markers for diagnosis,treatment,and prognosis assessment of nasopharyngeal carcinoma. |
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