Study Of Expression And Mechanism Of Major Facilitator Superfamily Domain Containing 4A In Nasopharyngeal Carcinoma

Objective: Nasopharyngeal carcinoma(NPC)is one of the most common malignancies.It has a high incidence in Southeast Asia and North Africa(especially in Southern China).With the development of intensitymodulated radiotherapy techniques from two-dimensional to widely used intensity-modulated radiotherapy,and the treatment from radical radiotherapy-based to the current integrated radiotherapy-chemotherapy treatment model,the diagnosis and five-year survival rate of NPC have been significantly improved.However,there are still 20%-30% of patients with residual cervical lymph nodes,recurrence of local tumor lesions or distant metastases after treatment with standard regimens,which become the main reason of treatment failure for NPC.Investigating the molecular mechanisms of NPC development,finding new therapeutic targets and related prognostic molecular indicators have become a challenge and opportunity to improve the survival rate of NPC.The major facilitaor superfamily(MFS)is one of the largest known families of membrane transporter proteins,which are closely related to viral,bacterial,drug resistance,autosomal recessive diseases,and anticancer issues.Major facilitator superfamily domain containing 4A(MFSD4A),a member of MFS,has been rarely reported for its expression and role in NPC.Our study intends to investigate the expression,biological function,mechanism of action and prognostic value of MFSD4 A in NPC.Methods:1.To investigate the methylation status of the promoter region of MFSD4 A and the expression of MFSD4 A in NPC tissues and cell lines and the regulatory relationshipThe methylation status of the MFSD4 A promoter region was first analyzed using multiple datasets in the public database GEO,and then the NPC tissues and NPC cell lines were collected to detect and validate the methylation of the MFSD4 A promoter region by pyrophosphate sequencing experiments.The expression levels of MFSD4 A in normal and nasopharyngeal cancer tissues,common nasopharyngeal epithelial cells and nasopharyngeal cancer cell lines were detected by Real Time Quantitative PCR and Western Blot(WB)to explore the relationship between the expression levels of MFSD4 A and the methylation status of the promoter region.2.To investigate the anti-cancer effect of MFSD4 A in NPC in vivo and in vitroIn vitro,MFSD4 A overexpression plasmid and small interfering RNA(si RNA)were constructed to transiently transfect HONE1 and SUNE1 cells respectively to increase or decrease the expression level of MFSD4 A in NPC cell lines.CCK8 cell proliferation assay,plate clone formation assay,cell migration and invasion assay were performed to examine the effects of MFSD4 A expression level on the proliferation,migration and invasion ability of NPC cells,respectively.In vivo,MFSD4 A stably overexpressed NPC cell lines were first constructed using a lentiviral vector system,and NPC cells stably overexpressing MFSD4 A were injected into the subcutaneous or tail vein of nude mice to construct subcutaneous transplantation tumor and lung metastasis tumor models.The effect of MFSD4 A expression level on the volume,tumor,HE staining,and immunohistochemistry of transplanted and metastatic tumors in nude mice was observed to investigate the effect of MFSD4 A on the proliferation and metastatic ability of nasopharyngeal carcinoma cells in vivo.3.To explore the molecular mechanism of the anti-cancer effect of MFSD4 A in NPCTranscriptome sequencing and mass spectrometry analysis were performed for MFSD4 A stably overexpressed NPC cell lines.The downstream signaling pathways and functional targets were screened by bioinformatics analysis,and then validated by real-time fluorescence quantitative PCR,protein immunoblotting,immunoprecipitation(CoImmunoprecipitation,Co-IP),and immunofluorescence(Immunofluorescence).Finally,we further clarified the biological functions and alterations of downstream signaling pathways mediated by MFSD4 A through downstream targets in NPC by functional backfill assays.4.To explore the prognostic value of MFSD4 A and its downstream in NPCWe collected paraffin tissue specimens from several patients with NPC for immunohistochemical analysis of MFSD4 A and its downstream,and investigated the correlation between the expression of MFSD4 A and its downstream;We performed risk stratification for patients with NPC based on immunohistochemical scores and analyzed the prognostic differences between patients in different risk groups.Results:1.In the tissue and cell lines of NPC,the reduced expression of MFSD4 A was associated with the hypermethylation status of its promoter region.2.MFSD4 A inhibited the proliferation,migration and invasion of NPC cells.3.MFSD4 A degraded EPHA2 by recruiting RNF149 and inhibited the downstream PI3K-AKT-ERK1/2 signaling pathway and epithelialmesenchymal transition(EMT)thus played an anti-cancer role.4.MFSD4 A and its downstream EPHA2 were independent prognostic factors for NPC.Patients with high MFSD4 A expression had better overall survival,disease-free survival,and distant metastasis-free survival;patients with high EPHA2 expression had poorer overall survival,diseasefree survival,local area recurrence-free survival,and distant metastasisfree survival.Conclusions:1.Hypermethylation of the promoter region of MFSD4 A leads to its reduced expression in nasopharyngeal carcinoma.2.MFSD4 A inhibits the growth,invasion and metastasis of nasopharyngeal carcinoma cells in vitro and in vivo through inhibition of PI3K-AKT-ERK1/2,and inhibition of epithelial-mesenchymal transition(EMT)process.3.MFSD4 A can recruit RNF149 to degrade EPHA2,thereby inhibiting its downstream PI3K-AKT-ERK1/2 signaling pathway and EMT.4.The protein expression levels of MFSD4 A and EPHA2 have prognostic significance.Patients with high MFSD4 A expression have a better prognosis.Patients with high EPHA2 expression have a poorer prognosis.Both MFSD4 A and EPHA2 are independent prognostic factors for nasopharyngeal carcinoma.