Low Expression Of 3-hydroxybutyrate Dehydrogenase Type 2 Promotes Proliferation And Metastasis Of Nasopharyngeal Carcinoma By Interfering Iron Homeostasis

Nasopharyngeal carcinoma(NPC)is a primary malignant tumor arising from the epithelium of nasopharynx.Most carcinoma site located at the top of nasopharynx and neighboring structures.In southern China,NPC is one of the high incidence of malignant tumors with distinct geographical,racial and ethnic distribution.The incidence of NPC is 15-30/100,000 people in southern China.The etiology of NPC is complicated.At present,three major etiological factors are involved in the carcinogenesis of NPC,including Epstein-Barr virus infection,genetic susceptibility,environmental factors.Nasopharynx is a complex anatomical structure with a lot of vital organs surrounded.Since the tumors are moderately radiosensitive,radical radiation therapy is the first choice of treatment.In recent years,with the development of diagnosis,radiotherapy techniques,and m?ltidisciplinary treatment,the efficacy of NPC therapy has been improved.The main reason of treatment failure in nasopharyngeal carcinoma is distant metastasis and local recurrence.So elucidating molec?lar mechanisms of NPC metastasis and searching for effective target therapies are the promising pathway to improve the survival.Iron participate in various kinds of metabolic processes,including DNA synthesis,Oxygen transport,KREBS CYCLE and facilitate the cell proliferation.The tumor cells can reg?late the expression of iron-related proteins to improve the level of iron intake,decrease the level of storage iron.These facors are better able to maintain tumor cells growth.3-hydroxybutyrate dehydrogenase type 2(BDH2)located on the human chromosome 4q24,encodes a member of the short-chain dehydrogenase/reductase family.As a key emzyme in ketogenesis,BDH2 mediates the first step of ketone body degradation between 3-hydroxybutyrate and acetoacetate in liver.BDH2 also manip?lates intracell?lar iron homeostasis by containing an iron-responsive element and mediating cell?lar iron tracking by catalyzing the synthesis of the mammalian siderophore that binds labile iron.With the deveploment of Deferoxamine in clinical theraphy,iron metabolism is a target for anticancer theraphy has been verified.Here,we explored the expression of BDH2 in NPC and addressed its effect on malignant behavior by reg?lating iron metabolism.ObjectiveTo investigate the abnormal expression of BDH2 and its malignant phenotypes in NPC.To explore reg?lation of BDH2 on the iron homeostasis and further analyzed the molec?lar mechanisms between iron homeostasis and malignant phenotypes in NPC.Methods1.Detecting the mRNA and protein level of BDH2 in nasopharyngeal carcinoma cells by Real time RT-PCR,and verifying nasopharyngeal carcinoma tissues by Western blot and immunohistochemistry assays.2.Establishing BDH2 stably overexpressed NPC cells by liposomes mediated gene transfection,and investigating the proliferative capability of BDH2 gene in NPC cells by MTT assay,colony formation assay,nude mouse tumorigenicity assay and wound healing and transwell assays.3.Detecting the differentially expressed genes in stable overexpression BDH2 NPC cells and the control cells by microarray.Analyse the sequencing data by GCBI(Gene Cloud of Biotechnology Information)analysis platform.4.Verifying the invasive ability of BDH2 gene in NPC cells by wound-healing assay and transwell assay.In order to reveal the molec?lar mechanisms,we perform western blot to detect the expression of E-Cadherin,Vimintin,?-Catenin,SPARC,EPCAM,which belong to invasion and metastasis.5.To confirm the effect of iron level in NPC.We investigated the level of iron in stable overexpression BDH2 NPC cells by Iron Colorimetric Assay Kit.NPC cells were co-c?ltured in different concentration ratio with deferasirox or iron(II)s?lfate heptahydrate.Then detect the invasive ability and proliferative capability by MTT assay,wound-healing assay and transwell assay.Results1.The m RNA and protein level of BDH2 were downreg?lated in NPC cells and tissuesWe detected the transcription of BDH2 in NPC cells and normal nasopharyngeal epithelium(NNE)cells by Real time RT-PCR.Our result showed that the transcription of BDH2 was downregulated in NPC cells.We further explore the expression of BDH2 in NPC and NNE biospies by Real time RT-PCR and immunohistochemistry staining assay.The results showed the expression of BDH2 was downregulated in NPC.2.BDH2 inhibits the proliferation of NPC cellsEstablishing the NPC cell line overexpressed BDH2,we found that ectopic expression of BDH2 inhibits the proliferation by MTT assay,colony formation,and nude mouse tumorigenicity assay.These suggest that BDH2 can inhibits the proliferates ability in NPC cells.migratory and invasive capacity of NPC cells.We found the expression of Snail,MMP9 and MMP2 were downreg?lated in BDH2 overexpressed NPC cells,while E-cadherin was upreg?lated,which indicating the role of BDH2 in reversing EMT in NPC.3.BDH2 suppresses the metastasis of NPC cells by reg?lating the gene expression related to tumor metastasisBy microarray technique,compare with control cells,we found the expression of E-Cadherin,EPCAM were upreg?lated in overexpressed BDH2 NPC cells,and Vimintin,?-Catenin,SPARC were downreg?lated.We also confirm that overexpression of BDH2 in NPC cells inhibites invasion by wound healing assay and transwell assay.We perform Real time RT-PCR and Western Blot to explore the expression of E-Cadherin,EPCAM,?-Catenin,SPARC.The res?lts were consistent with the microarray.4.BDH2 decreased cellular iron to inhibit the proliferation,migration and invasion of NPC cellWe found the iron level was decreased in NPC cells overexpressed BDH2.And the proliferation of NPC cells varies inversely with the DFX concentration.Iron supplement restored the proliferation of NPC cells and also promoted migration and invasion.These suggest that BDH2 decreased iron level to inhibit NPC proliferation,migration and invasion.Conclusion1.BDH2 was downreg?lated in NPC.2.Overexpression of BDH2 inhibits proliferation and invasion of NPC cells.3.BDH2 suppresses proliferation,migration and invasion of NPC cells by reduce iron content.Overall,BDH2 may be a candidate tumor suppressor gene in NPC.