Screening Of Radioresistance Genes SIVA1 And UBE2A In Nasopharyngeal Carcinoma And Their Correlation With Clinical Prognosis

[Objective]Radiation resistance of nasopharyngeal carcinoma can easily cause tumor residue,which leads to recurrence and metastasis is the main reason for the failure of nasopharyngeal carcinoma treatment.Therefore,this study screened out genes related to nasopharyngeal carcinoma radioresistance,and explored the molecular mechanism of radioresistance and the correlation between its expression and the clinical prognosis of nasopharyngeal carcinoma after radiotherapy,which can guide clinical individualized treatment and reduce nasopharyngeal carcinoma disease.Burden and improve survival have certain clinical significance.[Methods]1.Construct the nasopharyngeal carcinoma radiotherapy resistant cell line C666-1-IR,conduct cell migration,invasion,and apoptosis experiments,and use the transcription factor reporter gene system to find that p53 in C666-1 has a high background activity,and the activity of p53 in C666-1-IR is reduced,combined with biosynthesis data analysis and gene chip data（GSE32389）,the genes SIVA1 and UBE2A,which are closely related to p53 and affect the sensitivity of radiotherapy are screened.At the same time,the levels of SIVA1 and UBE2A m RNA in head and neck squamous cell carcinoma（HNSCC）in the TCGA database were collected for Kaplan-Meier survival analysis.2.The 52 newly diagnosed nasopharyngeal carcinoma patients who were admitted to the First Affiliated Hospital of Xiamen University from June 2016 to December 2018 were selected as the research subjects.The follow-up period ended in December 2020.Immunohistochemical experiments were performed to detect the protein levels of SIVA1 and UBE2A in the samples of patients with nasopharyngeal carcinoma before radiotherapy.Two pathologists scored the protein expression levels according to the corresponding standards.At the same time,the clinical characteristics and prognosis data of patients with nasopharyngeal carcinoma were collected.3.Chi-square test,Kaplan-Meier survival analysis and COX risk regression model were used to analyze the relationship between protein expression levels and clinical parameters and prognosis.[Results]1.Nasopharyngeal carcinoma resistant cell line enhances the ability of invasion and anti-apoptosis.The nasopharyngeal carcinoma cell line C666-1-IR group had no difference in cell migration ability compared with the C666-1 group（P>0.05）,but the invasiveness was enhanced（P<0.001）.Apoptosis experiments showed that the rate of apoptosis in the C666-1-IR group was lower than that in the C666-1 group（0.377%vs.5.32%;P<0.05）.2.Screening of radiotherapy resistance genes for nasopharyngeal carcinoma.The results of the transcription factor reporter gene system found that the p53 level in the cell line C666-1-IR group was lower than that in the C666-1 group.The C666-1-IR group and C666-1 group m RNA were transcriptome sequenced,and GO enrichment analysis of the differential genes showed that the“Negative Regulation of Signal Transduction by p53 Class Mediator”mediated by p53 in the C666-1-IR group,"Negative Regulation of Intrinsic Apoptotic Signaling Pathway in Response to DNA Damage"and"Negative Regulation of Apoptotic Signal Pathway"are inhibited.At the same time,combined with the GEO database gene chip（GSE32389）nasopharyngeal carcinoma radiotherapy sensitive and resistant tissue data,the differential genes SIVA1 and UBE2A were screened out.Using the HNSCC data in the TCGA public database to further analyze,the 3-year overall survival（OS）of patients with high SIVA1 m RNA expression was significantly lower than that of patients with low SIVA1 m RNA expression（P<0.001）;the 3-year OS of patients with high UBE2A m RNA expression was significantly lower than UBE2A m RNA.Patients with low expression（P=0.02）.3.The correlation between the expression of SIVA1 and UBE2A and clinical prognosis.Through immunohistochemistry at the protein level,SIVA1 and UBE2A proteins are both expressed in the nucleus and cytoplasm of nasopharyngeal carcinoma.Among 52 samples,SIVA1 protein was expressed in more than half of nasopharyngeal carcinoma tissues（31/52）,while UBE2A was generally expressed in tumor tissues（50/52）.Chi-square test analysis of SIVA1 protein expression level was significantly correlated with age（χ2=8.985,P=0.003）.UBE2A protein expression and age（χ2=5.039,P=0.025）,WHO classification（χ2=10.703,P=0.005）,N stage（χ2=6.002,P=0.014）,serum EBV DNA level（χ2=4.302,P=0.038）Significantly correlated.Compared with patients with SIVA1^negative or UBE2A^low expression in tumor cells,patients with SIVA1^negative expression or UBE2A^low expression had a significantly higher rate of radiotherapy resistance（P=0.012;P=0.0165）,and a higher proportion of disease recurrence or metastasis（P=0.014;P<0.001）,progression-free survival（PFS）decreased（HR =3.984[1.356-11.706],P<0.001;HR=4.983[1.762-14.088],P<0.001）.The proportion of patients with SIVA1^positive+UBE2A^high co-expression of tumor cells was higher than that of SIVA1^positive+UBE2A^low expression/SIVA1^negative+ UBE2A^highexpression group and SIVA1^negative+UBE2A^low expression group at 6 months after radiotherapy（P=0.017）,and disease occurred the risk of recurrence or metastasis（P<0.001）was higher,and PFS was reduced（P<0.001）.[Conclusion]SIVA1 and UBE2A are not only potential biomolecular markers for nasopharyngeal carcinoma resistance to radiotherapy,but also may have predictive significance for the clinical efficacy and disease progression of patients after radiotherapy.The expression of SIVA1^positive and UBE2A^high at the first diagnosis can effectively predict the radiotherapy resistance of patients with nasopharyngeal carcinoma.It is of clinical significance to detect the expression levels of the radiotherapy resistance genes SIVA1 and UBE2A in nasopharyngeal carcinoma by pre-treatment specimens,and to screen people who are resistant to radiotherapy and prone to disease progression,and achieve individualized treatment,reduce the burden of nasopharyngeal cancer and reduce mortality.