| Background and ObjectiveNasopharyngeal carcinoma(NPC)is a kind of head and neck malignant tumor in southeast Asia and China,which originates from the nasopharyngeal epithelium and has obvious genetic characteristics.75%to 90%of patients with NPC have locally advanced cervical lymph node metastasis at the time of initial treatment,and about 4.4%to 10%of patients have distant metastases,with a median survival of only 10 to 15 months.There is a lack of effective clinical treatment for rapid malignant proliferation and tumor growth of NPC,which has led to their becoming a difficult treatment point for NPC and a major cause of death for patients.At present,"metabolic reprogramming" is a major hot spot in the field of basic tumor research,and tumor cells are often accompanied by multiple metabolic pathway disorders,resulting in abnormal activation or instability of the cell state.In most tumor cells,glutamine metabolism is an important energy-producing metabolic pathway second only to glucose metabolism in cellular energy pathways,and is a particularly critical factor and link for the proliferation and growth of tumor cells.Glutaminase(GLS),one of the key enzymes in the glutamine metabolic pathway,has been confirmed in published studies that GLS can promote the migration,invasion,metastasis,proliferation and other biological functions of a variety of malignant tumor cells such as melanoma,pulmonary small cell lung cancer,triple-negative breast cancer and colorectal cancer,but GLS has not been published in the relevant studies and literature in NPC.Whether GLS has a malignant cancer-promoting phenotype similar to that in other tumors in NPC and the molecular mechanism of its effect need to be experimentally verified.The GLS inhibitor CB-839 has entered phase Ⅲ clinical trials for breast cancer,and its biological function and effect on NPC cells also need to be explored,which may provide new ideas and potential therapeutic targets for clinical diagnosis and treatment.Methods1.GLS expression levels at mRNA and protein levels were measured using immortalized nasopharyngeal epithelial cell line NP69 and five NPC cell lines.The nasopharyngeal tissue samples of 110 patients with NPC and 40 patients with nasopharyngitis were stained with immunohistochemistry to analyze the correlation with stage and prognosis.2.Using CCK-8,plate cloning,flow cytometry,transwell migration and boyden invasion assay tested NPC cell lines 5-8F/shGLS and CNE1/shGLS with stable GLS knockdown expression in vitro.The effect of GLS on NPC in vivo was examined by subcutaneous tumor formation assay in nude mice.To verify the regulation of GLS on proliferation,cell cloning,cell cycle,migration and invasion phenotypes of NPC cells.3.Using PCR Array microarray,the downstream CCND2 affected by GLS cell cycle disorder was screened out.The changes of related proteins,proliferation and cell cycle were determined by positive and negative reversal experiments to verify the existence of PI3K/AKT/mTOR pathway between them.4.NPC cell lines stimulated by CB-839 were used to verify the protein level,proliferation,cell cloning,cell cycle,migration and invasion.Result1.GLS is more highly expressed in nasopharynx than in nasopharyngitis,and its expression level is positively correlated with clinical stage and survival time.2.Interference with GLS expression can significantly inhibit the proliferation,clone forming ability,migration and invasion of NPC cells,and GLS inhibitor CB-839 has a similar inhibitory effect on NPC cells.3.Interference with GLS expression can inhibit the downstream PI3K/AKT/mTOR pathway,thereby inhibiting the expression of CCND2,ultimately leading to G1 phase arrest of the cell cycle and affecting the proliferation of NPC cells.ConclusionGLS,which are highly expressed in nasopharyngeal carcinoma,affect CCND2 expression by activating PI3K/AKT/mTOR pathway,thereby regulating cell cycle to promote cell proliferation and play the role of candidate oncogenes. |
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