The Role And Mechanism Of WWOX In Inhibiting The Growth Of Nasopharyngeal Carcinoma Cells By Regulating ERMAP Gene

?Objective?Nasopharyngeal carcinoma(NPC)is a malignant head and neck tumor that often occurs in southern China.The incidence of nasopharyngeal carcinoma is the 18 th in the world.Nasopharyngeal carcinoma(NPC)is characterized by regional centralization,complicated pathogenesis,hidden incidence,high metastatic and invasive ability,and so on.The occurrence and development of NPC involve the participation of multiple genes,reveal the pathogenesis of NPC,and search for related new oncogenes.Exploring tumor suppressor genes and obtaining new therapeutic targets are of great significance for early diagnosis and treatment and prognosis of nasopharyngeal carcinoma(NPC).WWOX,a novel tumor suppressor gene,was first discovered in 2000.It contains two WW domains and one redox structure.The WW domain can regulate the interaction between proteins and proteins.In the previous study,we found that the expression level of WWOX gene in nasopharyngeal carcinoma tissues decreased,and gradually decreased with the progression of the disease,which was associated with lymph node metastasis.However,WWOX was found to beinvolved in the occurrence and development of nasopharyngeal carcinoma.The role of metastasis and prognostic value remains unclear.The aim of this study is to investigate the role of WWOX in the carcinogenesis and development of nasopharyngeal carcinoma(NPC)and the downstream gene mechanism regulating malignant biological behavior of nasopharyngeal carcinoma(NPC).?Method?1.A lentivirus vector containing WWOX gene was designed and packaged and transfected into nasopharyngeal carcinoma cell line CNE-2Z to construct stable transfected cells.Affymetrix gene chip was used to detect the difference of human genome expression profile after overexpression of WWOX in nasopharyngeal carcinoma cell line CNE-2Z,and the significance of the differentially expressed genes was analyzed by using bioinformatics analysis software IPA,GO,et al.The enrichment analysis of classical signal pathway and classification of disease and function were carried out.The possible regulatory pathways and differentially displayed genes were screened to reveal the possible molecular mechanism of WWOX gene in nasopharyngeal carcinoma(NPC).2.On the basis of the suspected WWOX downstream nasopharyngeal carcinoma related genes found by Affymetrix gene chip,the upstream and downstream relationships between the candidate genes and WWOX were confirmed by RT-PCR method,and the downstream candidate genes were screened out again.Three candidate gene interference target sequences were designed,and the transfected cells were pre-mixed.The cell growth was monitored by Celigo full-field cell scanning analyzer in real time.The downstream driving genes which had the most significant influence on cell growth were analyzed and counted.Celigo was used for single target identification of candidate genes for follow-up experiments.3.RNAi lentivirus was prepared according to the sequence of downstream candidate genes identified by a single target,and stable transfected cell lines were constructed by infecting cells.The mRNA and protein levels of candidate genes were detected by Real-time PCR and Western blot,and the gene knockout efficiency was evaluated.The growth of down-regulated candidate genes was monitored by Celigo full-field cell scanning analyzer in real-time.The ability of cell proliferation and independent survival of down-regulated candidate genes were detected by clone formation assay.MTT assay was used to detect the effect of down-regulated candidate genes on cell proliferation.Flow cytometry was used to detect the effect of down-regulated candidate genes on early apoptosis.Transwell assay was used to detect the effect of down-regulated candidate genes on cell migration and invasion.4.A total of 81 nasopharyngeal tissues from patients undergoing nasopharyngeal surgery and 40 normal nasopharyngeal epithelial tissues from endoscopic sinus surgery and tonsillectomy were collected.Real-time PCR and immunohistochemical staining were used to compare the expression of candidate genes in nasopharyngeal carcinoma tissues and normal nasopharyngeal tissues,and the expression of candidate genes in nasopharyngeal carcinoma tissues was compared with that in normal nasopharynx tissues.The relationship between the expression of candidate gene protein and clinicopathological characteristics of nasopharyngeal carcinoma(NPC)was studied.?Results?1.After overexpression of WWOX,1112 differentially expressed genes were screened by microarray,of which 772 genes were up-regulated and340 genes were down-regulated.Pathway enrichment analysis showed thatdifferential genes mainly affected the state of several classical signaling pathways,including Role of BRCA1 in DNA Damage Response,Estrogen-mediated S-phase Entry,Protein Kinase A Signaling pathway.Differential genes play a major role in diseases and functions such as proliferation of cells,Viral Infection and organismal death.Further analysis of IPA,GO bioinformatics revealed that the differentially expressed genes involved in the biological processes of multiple malignant phenotypes and were involved in the genesis and development of tumors.2.On the basis of analyzing the results of a large number of gene data obtained by gene chip,the bioinformatics method is used to screen genes that have not been reported in nasopharyngeal carcinoma,which may affect the proliferation of tumor cells as the screening condition,and the method of bioinformatics is used to screen the genes that have not been reported in nasopharyngeal carcinoma.By sequencing the down-regulated gene P-value obtained by gene chip,30 candidate genes were selected.The expression of candidate genes was detected by RT-PCR in the cell lines stably transfected with WWOX gene,and the downstream genes were observed.Seven down-regulated candidate genes were obtained,so the functional screening of seven downstream candidate genes was carried out.Three interference targets were designed for each downstream candidate gene to obtain the mixed target RNAi lentivirus,which was transfected into CNE-2Z cells to ensure the interference efficiency.The proliferation of CNE-2Z cells was significantly affected by ERMAP gene in seven candidate genes by real-time detection with Celigo full-field cell scanning analyzer.The interference sequence shERMAP(55086),which has the most significant knock-down efficiency,was further verified by Celigo in order to prepare for follow-up experiments.3.The expression of ERMAP mRNA and protein in nasopharyngeal carcinoma cell line was relatively high,and the expression level of mRNA and protein in NPC cell line was significantly down-regulated after ERMAP knockdown by RNAi lentiviral knockout.Real-time monitoring of cell proliferation by Celigo showed that the number of cells in control group was6623 ±407.The number of cells in interference group was 682 ±36 on the 5th day of culture.After down-regulation of ERMAP,the ability of cell proliferation was weakened.Cell cloning experiment showed that the number of clones in control group and interference group was 188 ±4 and 20 ±1 respectively after 15 days of culture,and ERMAP knockdown inhibited cell clone formation.MTT assay showed that the proliferation multiple of the control group was 7.603±0.1471,and the interference group was 2.197 ±0.1579.The proliferation activity of CNE-2Z was significantly inhibited in the control group(7.603±0.1471)and the interference group(2 ±0.1579).The results of flow cytometry showed that knock-down of ERMAP could significantly increase the apoptosis rate of cells.Transwell assay showed that the migration ability of the cells was significantly inhibited.4.The expression of ERMAP mRNA in normal nasopharyngeal tissue and nasopharyngeal carcinoma tissue was 0.3395 ±0.188 and 1.050 ±0.609,respectively.The expression of ERMAP mRNA in nasopharyngeal carcinoma tissue was significantly higher than that in normal nasopharyngeal tissue(P<0.05).The average score of ERMAP immunohistochemical results was 5.63±2.47 in 40 normal nasopharyngeal tissues and 8.28 ±3.50 in 81 nasopharyngeal carcinoma tissues.The expression of ERMAP protein in nasopharyngeal carcinoma tissues was significantly higher than that in normal nasopharyngeal tissues.The difference was statistically significant.The relationship between theexpression of ERMAP and the clinicopathological features of nasopharyngeal carcinoma(NPC)was quantitatively analyzed.The results showed that the expression of ERMAP was correlated with clinical stage.The expression of ERMAP in patients with stage ? and ? was higher than that in patients with stage III,IV.The expression of ERMAP was significantly correlated with sex,age,histopathological type and T stage of nasopharyngeal carcinoma.There was no significant correlation between lymph node metastasis and lymph node metastasis(P > 0.05).?Conclusion?1.The function of WWOX may be accomplished by three classical tumor-related pathways.ERMAP,a downstream driving gene of WWOX,plays an important role in promoting tumorigenesis in nasopharyngeal carcinoma(NPC)cells,and this study provides a new target and idea for targeted therapy of nasopharyngeal carcinoma(NPC).2.Down-regulation of ERMAP gene can significantly reduce the proliferation,clone formation and migration ability of nasopharyngeal carcinoma cells,and down-regulate the expression of ERMAP plays an important role in reducing malignant phenotype of nasopharyngeal carcinoma.3.The expression of ERMAP in nasopharyngeal carcinoma was significantly higher than that in normal nasopharyngeal tissues,and may be related to TNM stage.ERMAP may play an important role in the carcinogenesis and development of nasopharyngeal carcinoma.