SOX2 Promotes Nasopharyngeal Carcinoma Cell Proliferation And Cisplatin Resistance By Up-Regulating The Transcription Level Of ZIC5

Background: Nasopharyngeal carcinoma(NPC)is one of the most common head and neck malignancies in southern China which incidence is closely related to genetics,Epstein-Barr virus(Epstein-Barr virus)infection,and poor living habits.There were about 129,000 new cases of nasopharyngeal cancer over the world,accounting for only about0.7% of all types of cancer in 2018,but its distribution has obvious regional and gender differences,about 70% occurred in East Asia and Southeast Asia.The treatment efficiency and survival rate of early nasopharyngeal carcinoma are relatively high,the treatment regimen of concurrent chemoradiotherapy and induction chemotherapy for locally advanced nasopharyngeal carcinoma had also made great progress recent years.However,recurrence and lymph node metastasis caused by cisplatin resistance may still lead to poor prognosis,so it is necessary to further clarify the molecular mechanism of NPC proliferation,migration and cisplatin resistance.ZIC5(Zinc finger protein of the cerebellum 5)is a member of the cerebellar zinc finger protein family which has been proved to play an important transcriptional regulatory role in embryonic development,especially in the process of neural differentiation.ZIC5 expression of malignant tumors including non-small cell lung cancer,malignant melanoma,and colorectal cancer has been proved to be elevated which participate in the regulation of biological functions such as proliferation and migration,but there is no research on the function and mechanism of ZIC5 in nasopharyngeal carcinoma.SOX2(SRY-box transcription factor 2)belongs to the SOX family B1 group with an HMG domain which recognizing and binding specific DNA consensus motifs as well as performing nuclear localization,while the transactivation domain can recognize and bind to the target promoter sequence of downstream gene to activate or repress its expression.SOX2 has been proved to play an important regulatory role in eukaryotic development,tumor formation and progression,and is involved in important processes such as stem cell reprogramming and cancer stem cell-like phenotype maintenance.Methods: First,by using R(v 3.6.3)and related software packages,the high-throughput sequencing data of 546 head and neck squamous cell carcinomas and normal tissue samples from The Cancer Genome Atlas(TCGA)were obtained,the gene chip data of 36 cases of nasopharyngeal carcinoma and normal tissues in the GSE53819 dataset of the Gene Expression Omnibus(GEO)was also acquired.Then differential analysis was performed to explore the expression and prognostic value of ZIC5 in nasopharyngeal carcinoma(NPC)and head and neck squamous cell carcinoma(HNSCC)comparing with normal tissues.Then,We applied quantitative Real-time PCR(qRT-PCR)and Western blot experiments to detect the m RNA and protein expression level of ZIC5 in NPC cell lines with different EBV-DNA carried state and differentiate level(HK-1,C666-1,CNE-1 and HNE-1)and compared with immortalized human nasopharyngeal normal cell line NP69.Next we chose C666-1 and HNE-1with high endogenous expression of ZIC5 to construct and validate stable silent ZIC5 cell line transfected by sh-ZIC5 lentivirus.CCK8 cell proliferation and toxicity experiments,scratch experiments,and colony formation experiments were conducted to clarify the gene function.Flow cytometry of cell apoptosis and cell cycle was also performed to further study the biological effect of ZIC5 on apoptosis ratio and cell cycle of nasopharyngeal carcinoma cell lines.Next we analyzed the expression level of SOX2 which is one of the stem cell markers and its correlation with ZIC5,then we speculated that SOX2 may have a regulatory relationship with ZIC5,further we predicted direct interaction between SOX2 with the promoter region of ZIC5 using the JASPAR database.Then we conducted chromatin co-immunoprecipitation and dual-luciferase reporter experiments to validate in vitro.Loss-of-function and rescue experiments mediated by lentivirus and overexpression plasmid vector were performed to determine the upstream and downstream relationship between SOX2 and ZIC5.Finally we used sh-ZIC5 HNE-1 cells to conduct subcutaneous tumorigenesis experiments in nude mice to validate the effect of ZIC5 on the proliferation and cisplatin sensitivity of nasopharyngeal carcinoma cells in vivo.Results: ZIC5 m RNA was highly expressed in primary nasopharyngeal carcinoma in GSE53819 dataset(Log2FC=2.900,p<0.0001),also highly expressed in head and neck squamous cell carcinoma in TCGA-HNSC data(p<0.001).However,there was no significant difference in the expression levels among patients with different clinical stages(Pr=0.379),and prognosis between the high and low ZIC5 expression group(p=0.17).We found that ZIC5 m RNA and protein were endogenously highly expressed in HNE-1 and C666-1nasopharyngeal carcinoma cell lines(all nasopharyngeal carcinoma cell lines were at least 1.47 times higher than NP69,p<0.01).ZIC5 m RNA and protein expression levels of HNE-1 and C666-1 cell lines transfected with sh-ZIC5 were significantly down-regulated(p<0.05),indicating that the stably transfected cell lines were successfully constructed.The proliferation and migration ability of ZIC5 knock-down cells was significantly decreased(All p<0.05),the proportion of apoptotic cells and the sensitivity to cisplatin were significantly increased(All p<0.05),the proportion of cells in G1 phase was significantly increased and cells in G2 phase was decreased which is G0/G1 phase cycle arrest(All p<0.05).There was no significant difference in the expression of SOX2 in primary nasopharyngeal carcinoma and head and neck squamous cell carcinoma compared with normal tissues(GSE: p=0.097,TCGA: p=0.53),but the expression of SOX2 and ZIC5 has certain weak correlation(GSE:p=0.021,R=0.385;TCGA: p=7.5e-21,R=0.4).We predicted SOX2 may directly bind to the ZIC5 promoter region using the JASPAR database,and performed chromatin immunoprecipitation experiment to prove the direct bind of SOX2 with ZIC5 promoter region(p<0.01),then we validated the result by dual luciferase reporter experiment in vitro(p<0.01).Function lost/rescue experiments showed knock down of SOX2 can reduce the proliferation,migration and resistance to cisplatin in nasopharyngeal carcinoma cells by downregulating ZIC5(All p<0.05),also lead to cell cycle arrest in G0/G1 and increased proportion of apoptotic cells(All p<0.05),most importantly the above biological function changes can be partially rescued by ZIC5 overexpression(All p<0.05)indicating that SOX2 is a direct upstream regulator of ZIC5.ZIC5 knockdown in vivo significantly inhibited the growth of xenograft tumors in nude mice(p<0.01),qRT-PCR and Western blot of the tumors confirmed no significant difference in SOX2 expression between different groups(p>0.05),and significantly down-regulated expression level of ZIC5(p<0.01)in sh-ZIC5 group,Ki67 immunohistochemical results of tumor tissue also showed ZIC5-knockdown significantly inhibit the proliferation activity of nasopharyngeal carcinoma cells in vivo(p<0.01).Conclusion: SOX2 promotes the proliferation,migration and cisplatin resistance of nasopharyngeal carcinoma cells by up-regulating the transcription level of ZIC5.