CCDC86 Promotes The Proliferation,Invasion And Migration Of Nasopharyngeal Carcinoma Cells Via Positively Regulating The EGFR-PI3K/Akt Signaling

Background and Objective:Nasopharyngeal carcinoma(NPC)is a malignant tumor originating from the nasopharyngeal epithelium.It has obvious regional and ethnic distribution,and has a high incidence in southern China and Southeast Asian countries.At present,the treatment of NPC is based on radiotherapy,combined with chemotherapy and some other modes such as surgical treatment,targeted therapy and immunotherapy.CCDC86(coiled-coil domain-containing protein 86)is a phosphorylated nucleoprotein,which has been proved by many studies to be related to the occurrence and development of lymphoma and is a relatively new tumor-related protein.However,the effect of CCDC86 on NPC has not been reported yet.In this study,in vivo and in vitro experiments were conducted to explore whether CCDC86 plays a role in the pathogenesis of NPC and to further explore its specific mechanism.It provides a new idea for studying the pathogenesis of NPC and discovering new drug targets.Methods:1.Clinical data of NPC patients were collected,and patients were followed up to obtain survival data.Immunohistochemistry(IHC)was used to detect the difference in the expression of CCDC86 in NPC tissues and normal nasopharyngeal epithelial tissues.2.The mRNA expression of CCDC86 gene in 7 NPC cell lines and 2 normal nasopharyngeal epithelial cell lines were analyzed by qPCR.3.CCDC86 knockdown and overexpression studies were performed in 2 NPC cell lines to evaluate changes in cell proliferation,migration and invasion by CCK8,EDU,clone formation technology,wound healing assay,and Transwell chamber test.4.The expression of EMT-related and MMPs proteins were detected by Werstern Blot in CCDC86 knockdown cells.5.In vivo experiment: Nude mice were injected CCDC86 knockdown cells through tail vein to establish a lung metastatic mice model and to study the characteristics of metastasis of NPC in vivo.6.Microarray and IPA analysis was used to analyze the network interaction map of CCDC86 related genes to search for downstream regulatory genes,and the protein and mRNA expression differences of downstream genes were detected by WB and qPCR.7.The changes in cell proliferation,invasion and migration ability were measured by CCK8,EDU and transwell invasion assays in cells with and without CCDC86 knockdown and the cells cultured with EGF.8.The expression of PI3 K,Akt protein and their phosphorylation levels were detected by Werstern Blot in cells with and without CCDC86 knockdown and the cells cultured with EGF.9.MS and Co-IP assays were used to analyze directly interacting proteins downstream of CCDC86.Results:1.Immunohistochemical(IHC)staining showed that CCDC86 was localized in the cytoplasm of cells and was highly expressed in the NPC tissues(n = 124).Survival curve analysis showed that the overall survival rate of NPC patients was not significantly different between the high CCDC86 expression group and the low expression group during the follow-up.However,the progression-free survival rate in the low CCDC86 expression group was higher than that in the high expression group,and it was statistically significant in the patients of stage ?-?.2.Except for HONE1 and HK1 cells,most of the NPC cell lines showed a higher mRNA level of CCDC86 as compared with NP69 cells and the difference was statistically significant.3.CCDC86 knockdown inhibits NPC cell proliferation,migration and invasion,while overexpression of CCDC86 promotes the proliferation,invasion and migration of NPC cells in vitro.4.Compared with the control group,the expression of E-cadherin in the experimental group was up-regulated and ?-catenin,Vimentin,MMP2 and MMP-9were down-regulated in CCDC86 knockdown cell lines,suggesting that the CCDC86 knockdown can inhibit the expression of MMPs and reverse the EMT process in NPC cell lines.5.In vivo experiment: CCDC86 knockdown inhibits the metastasis of NPC cells in vivo.6.The results of microarray and IPA assays suggest that CCDC86 plays a role in nasopharyngeal carcinoma by regulating the expression of EGFR,BCL2L11 and BIRC5 genes.In this study,EGFR was selected for subsequent experiments.The results of WB and qPCR confirmed that EGFR protein and mRNA expression were down-regulated in CCDC86 knockdown cell lines,and the difference was statistically significant.7.Combined with down-regulation of CCDC86 and up-regulation of EGFR,the cell function recovery experiment confirmed that CCDC86 promotes NPC cell metastasis by upregulating EGFR expression8.The results of WB detection of downstream signaling pathways confirmed that CCDC86 regulates EGFR to be involved in the PI3K/Akt signaling pathway.9.MS and Co-IP experiments confirmed that CCDC86 was not directly associated with EGFR,but mediated by binding with NPM1 to regulate EGFR.Conclusions:CCDC86 positively regulates the EGFR-PI3K/Akt signaling pathway through binding with NPM1,and ultimately promotes the proliferation,invasion and migration of NPC.The expression level of CCDC86 may be a prognostic indicator for patients with advanced nasopharyngeal carcinoma.Therefore,CCDC86 is expected to be a new biomarker and therapeutic target for NPC patients.