VPS33B Interacts With NESG1 To Inhibit Proliferation And Induce 5-Fluorouracil Sensitivity In Nasopharyngeal Carcinoma

Background and ObjectivesNasopharyngeal carcinoma(NPC)is a tumor stemming from the nasopharyngeal tissue.It is epidemic in certain regions of East Asia especially Guangdong and Guangxi provinces of China.NPC is highly malignant with early local or distant metastasis,while it is inclined to show radiotherapy resistance in middle or advanced stage.According to recent researches,Epstein-Barr virus(EBV)exposure,diet and genetic factors attribute to the carcinogenesis of NPC.In recent years,NPC is becoming more epidemic in the youth.NPC is sensitive to the radioactive rays that radiotherapy is the first choice to in the early stage.Unluckily,because of the regular radiotherapy resistance,adjuvant chemotherapy is urgent.Oncogenes are essential in the normal proliferation that has the potential to cause cancer.Tumor suppressor genes,or antioncogenes,protect a cell from activating to cancer.When these genes mutate to cause a loss or reduction in their functions,usually in combination with other genetic changes,the normal cells progress to cancer cells.The imbalance between oncogenes and antioncogenes contributes essentially to carcinogenesis of almost all kinds.The vacuolar protein sorting 33B(VPS33B)is a member of the Sec-1 domain family and encodes the human ortholog of rat Vps33b,which is homologous to the yeast class C Vps33 protein.Mutations in this gene are associated with arthrogryposis-renal dysfunction-cholestasis syndrome.Also,VPS33B plays a vital role in megakaryocyte biogenesis,platelet activation and in vivo thrombosis and hemostasis.In addition,VPS33B has been identified as a tumor suppressor in hepatocellular carcinoma(HCC).NESG1(also known as CCDC19),encodes the cilia and flagella associated protein 45,which is specially expressed in human nasopharynx and trachea.We cloned and revised its coding sequence and preliminarily confirmed it as a potential tumor suppressor in NPC and non-small cell lung cancer(NSCLC)based on previous studies.In this study,we confirmed that VPS33B is downregulated in the nicotine-treated and LMP-1-overexpressing NPC cells through targeting PI3K/AKT/c-Jun signaling.Moreover,the interaction of VPS33B with NESG1 suppresses the proliferation and the chemoresistance to 5-fluorouracil(5-Fu)in NPC by inactivating the EGFR/PI3K/AKT/c-Myc/P53/miR-133a-3p feedback loop mechanism.Our findings provide,for the first time,a deeper understanding of the mechanism of the antiproliferative effect of VPS33B on NPC cells.Contents and methods1.VPS33B modulated EGFR/PI3K/AKT signal and sensitized NPC cells to 5-Fluorouracil.Using MTT assay and nude mice survival model to examine the 5-Fu sensitivity of VPS33B-overexpressing NPC cells in vivo and in vitro.2.miR-133a-3p suppressed proliferation of NPC cells.(1)Using MTT assay and Edu incorporation assay to examine the effect of miR-133a-3p expression on NPC cells or NP cell growth and definite the function of miR-133a-3p in NPC cells;(2)To explore the mechanisms that miR-133a-3p suppresses NPC cell proliferation,Western blot was used to examine whether the expression of EGFR,p-PI3K,p-AKT,c-Myc,p53 were influenced by miR-133a-3p;(3)Luciferase Activity Report indicated that miR-133a-3p directly targeted EGFR.3.C-Myc/p53 inactivated the suppressive effect of miR-133a-3p in the proliferation of NPC cells via the modulation of the EGFR/PI3K/AKT signaling pathway(1)To test the transcriptional regulatory mechanisms of miR-133a-3p and p53 expression,online bioinformatics software was utilized to analyze a 2-kb region upstream of the transcription start site(TSS)of miR-133a-3p and p53.P53 and c-Myc were identified inside the putative miR-133a-3p and p53 promoter region;(2)Examining the expression of miR-133a-3p and p53 after overexpressing p53 and c-Myc in NPC cells by qPCR assay;(3)EMSA assay detected whether the three predicted p53 and c-Myc binding sites in the promoter region of miR-133a-3p and p53 were functional;(4)Chromatin immunoprecipitation(ChIP)assays further confirmed that p53 and c-Myc protein was recruited to the predicted binding sites in the putative miR-133a-3p and p53 promoter in NPC cells;(5)Luciferase indicated that p53 and c-Myc modulate the transcription activity of miR-133a-3p and p53 promoter respectively.(6)Using Western Blot to detect the expression of the changes of c-Myc/p53 in related signals.(7)Examining the expression of miR-133a-3p in c-Myc overexpressing NPC cells after inducing p53 by qPCR assay.4.miR-133a-3p participated in the VPS33B-modulated EGFR/PI3K/AKT/c-Myc/p53 signaling pathway and the formation of the loop(1)Detecting the expression of miR-133a-3p in VPS33B-overexpressing NPC cells by qPCR assay;(2)Using MTT and Edu assays to examine the proliferation of VPS33B-overexpressing NPC cells after inducing EGFR;(3)To explore the mechanisms that EGFR reverses the inhibitory effects of VPS33B in NPC cell proliferation,Western blot was used to examine whether the expression of EGFR/PI3K/AKT/c-Myc/p53 were influenced by inducing EGFR;(4)Detecting the expression of miR-133a-3p in VPS33B-overexpressing NPC cells after inducing EGFR by qPCR assay.5.VPS33B interacted with NESG1 and stimulated each other in NPC cells(1)Using qPCR and Western Blot assays to determine the mRNA and protein expression of NESG1 or VPS33B in VPS33B-overexpressing or NESG1-overexpressing NPC cells;(2)Demonstrating whether VPS33B interacts with NESG1 via CO-IP assay;(3)By checking online bioinformatics database,c-Jun was identified inside the putative VPS33B and NESG1 promoter region;(4)EMSA and ChIP assay further confirmed the effectiveness of the predicted binding sites of c-Jun with VPS33B or NESG1 promoter;(5)Luciferase indicated that c-Jun modulated the transcription activity of VPS33B and NESG1 promoter respectively;(6)Using MTT and Edu assays to examine the proliferation of VPS33B-overexpressing NPC cells after silencing NESG1;(7)Western blot was used to examine whether the expression of EGFR/PI3K/AKT/c-Jun were influenced by inducing EGFR in NESG1-overexpressing NPC cells.6.LMP-1 and nicotine suppressed VPS33B expression through PI3K/AKT/c-.Jun signaling.(1)Using qPCR and Western Blot to determine the expression of VPS33B after treatment of LMP-1 and different concentrations or time-related nicotine in NPC cells;(2)Demonstrating whether LMP-1 could reverse the inhibitory effects of VPS33B in NPC cells by MTT and Edu assays.;(3)Using Western Blot to detect the expression of EGFR/PI3K/AKT related signals after transfecting LMP-1 in NPC cells;(4)Examining the changes in PI3K/AKT/c-Jun after inducing Ly94002 in NPC cells treated with nicotine.7.The expression of VPS33B in NPC tissues and its clinical characteristics(1)Detecting the expression of VPS33B and the co-expression of VPS33B with NESG1 in NP and NPC tissues by IHC.(2)Analyzing the correlation of VPS33B with NESG1 and the clinical characteristics of VPS33BResults and Conclusions1.VPS33B modulated EGFR/PI3K/AKT signals to sensitize NPC cells to 5-Fluorouracil.2.miR-133a-3p suppressed the proliferation of NPC.3.c-Myc/p53 inactivated the suppressive effect of miR-133a-3p in the proliferation of NPC cells via the modulation of the EGFR/PI3K/AKT signaling pathway.4.miR-133a-3p participated in the VPS33B-modulated EGFR/PI3K/AKT/c-Myc/p53 signaling pathway and the formation of a loop.5.VPS33B interacted and stimulated with NESG1 through suppressing EGFR/PI3K/AKT/c-Jun pathway in NPC cells.6.LMP-1 and nicotine suppressed VPS33B expression through PI3K/AKT/c-Jun signaling.7.Low expression of VPS33B correlated with bad prognosis of NPC patients.