The Role Of MAZ Mediated Autophagy Inhibition In Nasopharyngeal Carcinoma Metastasis And The Underlying Molecular Mechanisms

Background:Nasopharyngeal carcinoma(NPC)is one of the most common malignant tumors of head and neck in China.Radiotherapy is the main treatment of nasopharyngeal carcinoma,with the advanced development of radiotherapy,local control rate has been increased significantly,but distant metastasis after radiotherapyremains the major failure pattern,our previous study also showed that distant metastasis was the main reason for failure of nasopharyngeal carcinoma undergoing radiotherapy.The mechanism of nasopharyngeal carcinoma metastasis is not clear,and most research was focused on the cell adhesion,apoptosis,angiogenesis-related molecules and EB V virus,etc.However,these molecules were still unable to fully explain the molecular mechanisms of nasopharyngeal carcinoma metastasis.Some studies have found that inhibition of autophagy could promote tumor metastasis(Nature,2011;Cancer cell,2014),but the relationship between autophagy and nasopharyngeal carcinoma metastasis has not yet been reported.The number of autophagic bubbles in patients with metastatic nasopharyngeal carcinoma was significantly reduced,suggesting that inhibition of autophagy might promote the metastasis of nasopharyngeal carcinoma.MAZ(Myc-associated Zinc-finger protein),a novel gene related to metastasis of nasopharyngeal carcinoma,was found by genome-wide expression screening in our previous study.Our study also suggested that MAZ might promote nasopharyngeal carcinoma metastasis through autophagy inhibition.The relationship between autophagy and nasopharyngeal carcinoma metastasis and the pathway in which MAZ was involved during the process of autophagy inhibition and nasopharyngeal carcinoma metastasis were the focus of this study.Some study found that c-Myc could inhibit autophagy by upregulating the expression of 4EBP1,while the classical autophagic pathway PI3K/AKT/mTOR could upregulate c-Myc to promote tumor metastasis.We also predicted that MAZ could bind to the promoter region of c-Myc by bioinformatics prediction analysis.Therefore,does MAZ inhibit autophagy by interacting with c-Myc? Does PI3K/AKT inhibit autophagy b y modulating MAZ and c-Myc? What is the relationship between mTOR and MAZ/ c-Myc? Based on preliminary work and literature review,we proposed a theoretical hypothesis that MAZ could promotes metastasisof nasopharyngeal carcinoma through inhibitingautophagy and,and MAZ might be regulated by PI3 K / AKT / mTOR signaling and interacted with c-Myc during this process.Objective:This research was to study the relationship between autophagy and nasopharyngeal carcinoma metastasis and the pathway in which MAZ was involved during the process of autophagy inhibition and nasopharyngeal carcinoma metastasis.This study might help to elucidate the molecular mechanisms of metastasisof nasopharyngeal carcinoma and provide a new possible target for the intervention of nasopharyngeal carcinoma metastasis.Methods:1.The cells were irradiated with different doses of X-rays and the appropriate irradiation dose was screened by CCK-8 method.The high metastatic potential cell line 5-8F and low metastatic potential cell line 6-10 B of nasopharyngeal carcinoma cell line SUNE1 were selected The irradiated nasopharyngeal carcinoma cells were used for further experiments.2.Real-time quantitative PCR and Western blot were used to detect the mRNA and protein expression levels of 5-8F and 6-10 B of autophagy and metastasis-related genes.The expression of MAZ gene was also detected.3.MAZ shRNA expression vector was stably transfected to nasopharyngeal carcinoma cells.The interaction between MAZ and c-Myc/mTOR was confirmed b y Co-IP method.The relationship between MAZ and mTOR was detected by Western blot.The relationship between MAZ and PI3K/AKT/mT OR signaling pathway was also detected by Western blot.4.Effects of MAZ,c-Myc gene and PI3K/AKT/mTOR signaling pathway inhibitors test by qRT-PCR,Western blot,Wound-healing assay,Transwell assay,immunofluorescence,laser confocal,transmission electron microscopy.Results:1.4 Gy was the optimal X-ray dose for subsequent experiments.2.Validation of the level of autophagy was inversely related with metastatic ability of nasopharyngeal carcinoma cells.3.Successful construction of MAZ down-regulated nasopharyngeal carcinoma cell lines,the results showed that MAZ inhibition could enhance cell autophagy and inhibit the metastatic and proliferate capacity of NPC cell lines..4.The interaction between MAZ and c-Myc inhibited the autophagy level of nasopharyngeal carcinoma cells and c-Myc inhibition could enhance autophagy level and inhibit the metastatic ability of NPC cell lines.5.MAZ interacted with mTOR.MAZ promoted metastasis of nasopharyngeal carcinoma cell lines through autophagy inhibition by mTORC1 pathway,a nd was regulated by AKT signal.Conclusions:1.The level of autophagy in nasopharyngeal carcinoma cells is closely related to the ability of metastasis.2.MAZ could promote nasopharyngeal carcinoma cell metastasis by inhibiting autophagy.3.MAZ interacted with mTOR.MAZ promoted metastasis of nasopharyngeal carcinoma cell lines through autophagy inhibition by mTORC1 pathway,and was regulated by AKT signal.