Inactivation Of HMGCL Promotes Proliferation And Metastasis Of Nasopharyngeal Carcinoma By Suppressing Oxidative Stress

Nasopharyngeal carcinoma(NPC)is a malignancy frequently originating in the slit-like nasopharyngeal recess called fossa of Rosenmüller.NPC is associated with distinct geographical,racial and ethnic distribution.The incidence of NPC is less than 1/100,000 people worldwide,but the cancer is endemic in Southeast Asia and southern China,with an incidence of 20 to30/100,000 people.Multiple factors are involved in the carcinogenesis of NPC,including genetic susceptibility,environmental factors and Epstein-Barr virus(EBV)infection.NPC is conventionally treated with radiotherapy and early-stage NPC can be cured this way,but a significant number of patients still show local recurrence and distant metastases,so novel strategies for NPC therapy are needed.Deregulating cellular energetics is a hallmark of cancer.Accompanied with the abnormal fatty acids metabolism,the ketogenesis might alter in tumor cells,which would further change its biological behavior.In previous study,we foundthat the cellular ketone bodies were elevated in nasopharyngeal carcinoma(NPC).Interestingly,we found HMGCL gene by bioinformatics analysis,involved in the ketogenesis,were inactivated in NPC.Further,we verified the role of HMGCL in inhibiting the proliferation and migration of NPC cells.Our data suggest that HMGCL might inhibit tumor proliferation and metastasis by regulating ketogenesis.Therefore,in this study,we will validate the inactivation of HMGCL gene in NPC,and their effect on the production of ketone body.Furthermore,we will explore the function of these two genes in the proliferation and metastasis of NPC cells,and molecular mechanisms involved.This study would shed a new light into the pathogenesis of NPC in term of ketone metabolism,and provide a new target for NPC treatment.Objective:To explore the abnormal expression of HMGCL and its’ function role in NPC.To investigate the regulation of HMGCL gene on the production of ketone bodies in NPC,and further analyzed the association between intracellular/extracellular β-HB and ROS level in NPC.Methods:1.Screening out the differentially expressed genes related to ketone metabolism in NPC by analyzing the expression profiles of NPC from GEO database of NCBI.2.Analyzing the expression of HMGCL gene in 11 types of human tumors based on the TCGA database.3.Verifying the transcription and expression of HMGCL in nasopharyngeal carcinoma was verified by real-time RT-PCR,Western blot and immunohistochemistry assays,respectively.4.Establishing HMGCL stably overexpressed NPC cells,and investigating the biological function of HMGCL gene in NPC cells by performing proliferation assay,colony formation assay,wound healing and transwell assays.Detecting the expression of epithelial mesenchymal transition(EMT)related factors E-cadherin,Vimintin,β-cetenin by Western blot assay.5.Detection the functional role of exogenous ketone body on cell malignant biological behavior by using MTT assay and wound healing assay;evaluating the influence of endogenous and exogenous ketones on ROS oxidative stress,and further use of ROS inhibitor NAC in NPC cells,whether HMGCL depend on the activation of ROS affects the proliferation of NPC cells and metastasis.Result:1.Bioinformatics analysis suggested that the expression of HMGCL gene was down regulated in NPC tissuesThe results showed that the expression of HMGCL was down regulated by2 fold(P<0.05)in nasopharyngeal carcinoma.This suggests that HMGCL gene may play a key role in the nasopharyngeal carcinoma abnormal ketone metabolism.2.Bioinformatics analysis showed that HMGCL gene was down regulated in multiple tumors.The results showed that HMGCL gene was expressed in 11 types of cancer,including colon cancer,lung cancer,head and neck squamous cell carcinoma,renal cell carcinoma,etc.;it is suggested that the down-regulation of HMGCL gene expression may be a common phenomenon in cancer cells,which may be related to the occurrence and development of human cancers.3.The expression of HMGCL is downregulated in NPC.As compared with an immortalized normal nasopharyngeal epithelial cellline(NP69),6 NPC cell lines showed reduced mRNA level of HMGCL.As well,the expression of the HMGCL gene was downregulated in NPC primary tumors but easily detected in all NNE samples.In addition,we measured the protein levels of HMGCL in NPC tissue by immunohistochemical(IHC)staining.HMCGL was localized in the cytoplasm of cells and was highly expressed in the NNE but was almost absent in NPC tissue.4.Exogenous expression of HMGCL gene in nasopharyngeal carcinoma cells significantly upregulation of endogenous β-hydroxybutyrate.Endogenous and exogenous β-hydroxybutyrate production is positively related to the level of ROS.We assessed the relative concentration of intracellular β-HB,a main component of ketone bodies in NPC cells.Intracellular β-HB level was significantly higher with HMGCL-HK1 than pCMV6-Entry-HK1 transfection.Further,we analyzed the association between intracellular/extracellular β-HB and ROS level.β-HB generated by ectopic expression of HMGCL as well as extracellular β-HB treatment elevated ROS production in NPC cells.5.Exogenous expression of HMGCL inhibits NPC cell migration and invasion by reversing the epithelial–mesenchymal transition(EMT)To investigate the role of HMGCL in the metastatic potential of NPC cells,we used 2D and 3D model systems to determine the capacity for migration and invasion,respectively.In scratch assay,the gap closure was slower for HMGCL-HK1/5-8F than pCMV6-Entry-HK1/5-8F cells,so HMGCL expression retarded the migration of NPC cells.Transwell assay also confirmed that HMGCL overexpression markedly inhibited the invasive capacity of HK1 and5-8F cells.To reveal the underlying mechanism of HMGCL,we evaluated its effect on key EMT-associated proteins.The expression of E-cadherin wasupregulated in HMGCL-HK1/5-8F cells and that of β-catenin and vimentin was decreased.Overexpression of HMGCL may reverse the EMT process,thereby inhibiting the metastasis potential of NPC cells.6.HMGCL increases intracellular β-hydroxybutyrate(β-HB)level and generates ROS in NPC cells,and ROS inhibitor promotes NPC cell growth and migration.We found that β-HB generated by ectopic expression of HMGCL as well as extracellular β-HB treatment elevated ROS production in NPC cells,suggesting that the effect of HMGCL expression in NPC cells may be mediated by ROS.Conclusion:1.The expression of HMGCL is downregulated in NPC.2.Exogenous HMGCL gene inhibits the malignant biological behavior of nasopharyngeal carcinoma cells.3.Endogenous and exogenous ketone bodies can inhibit the malignant biological behavior of nasopharyngeal carcinoma by regulating the level of ROS.In summary,HMGCL might be a candidate tumor suppressor gene in NPC.