Endogenous Hydrogen Sulfide Synthase CSE Regulates The Growth And Mechanism Research Of Human Nasopharyngeal Carcinoma Cells

BackgroundNasopharyngeal carcinoma(NPC)is a tumor of the head and neck.While the geographical distribution of the NPC worldwide is abnormally twisted,mainly occurring in Southeast Asia.The main pathogenesis includes genetic,environmental,Epstein-Barr virus(EBV)infection and other factors.In the clinical treatment,nasopharyngeal carcinoma is mainly intervened through radio chemotherapy and surgery Because of its easy recurrence,early metastasis and the treatment effect is not perfect.Therefore,searching for the mechanism of occurrence and development of nasopharyngeal carcinoma,therapeutic targets and biomarkers etc for more targeted and precise treatment has become the most important goal at presentHydrogen sulfide(H2S)is a water-soluble gas with the smell of rotten eggs.In the past few decades,due to its ability to inhibit the hydrogen sulfide respiratory complex in mitochondria,the cells are unable to use oxygen for oxidative metabolism,Therefore H2S has been considered to be unfavorable to the human body.In recent years,more and more evidence shows that hydrogen sulfide is involved in different physiological and pathophysiological processes,including hypertension,neurodegenerative changes,inflammation and metabolic syndrome.Similarly,H2S also plays an important role in the occurrence and development of tumors.It may be a new molecular target in the precise treatment of tumor treatment.Current research work showed that hydrogen sulfide has an inhibitory effect and a promoting effect in tumors.This phenomenon may be related to the concentration and the duration of hydrogen sulfide action.Studies have shown that low concentrations can produce a promoting effect,and high concentrations show an inhibitory effect.In different tumors,the expression of three kinds of hydrogen sulfide synthase is also different,different enzymes have different effects on the tumor.At present,there are no reports on H2S and nasopharyngeal carcinoma at national and internationally level.To understand the role of hydrogen sulfide endogenous synthesis of key enzyme--Cystathionine-γ-lyase(CSE)in the progression and outcome of human nasopharyngeal carcinoma.We studied the overexpression/knockdown of CSE in-vivo and in-vitro and their effects on the growth and apoptosis of human nasopharyngeal carcinoma cellsPurposeTo study the expression of hydrogen sulfide endogenous synthesis key enzyme CSE in human nasopharyngeal carcinoma cells and its effects on the proliferation,migration,invasion,cell cycle and apoptosis of human nasopharyngeal carcinoma cells CNE-1 and C666-1 and related mechanisms have been further studied.MethodsDetect the expression of CSE in human nasopharyngeal carcinoma cells by Western blot,RT-PCR and human hydrogen sulfide content kit.Once confirming the high expression of CSE in human nasopharyngeal carcinoma cells,overexpress and silence or knocked down the CSE,matching blank or control.The control plasmids were transfected into human nasopharyngeal carcinoma cell lines CNE-1 and C666-1 respectively,and the optimal G418 concentration was determined to select stable cell lines.A series of experiments such as MTS,EdU,Tunel staining,plate cloning,scratching,Transwell,soft agar colony formation,and Invasion were used to determine their proliferation,apoptosis,cloning,migration and invasion ability in-vitro.Cell flow cytometry and Western blot were used to detect cycle-related proteins during the experiments;protein immunoblot was also used to detect apoptosis and invasion-related protein expression and AKT/PI3K/mTOR,ERK/P38/JNK signals Pathways.While in-vivo experiments,using nude mice right forelimb xenograft tumor experiment to study the effect of CSE on the growth of transplanted tumors in-Vivo,and HE,KI67,CD31,MMP-9 and P21 immunohistochemically detection of tumor tissues to study their correlation effect.Results1.Western blot,RT-PCR and human hydrogen sulfide content kit results explained that,when compared normal human nasopharyngeal epithelial cells with human nasopharyngeal cancer cells,the CSE expression are high in nasopharyngeal cancer cells2.The results of cell proliferation experiments MTS and EDU show that the CSE overexpression group can promote cell growth,although opposite for silencing or knockdown3.The results of scratches and Transwell in the experiment of measuring cell migration ability show that the CSE overexpression group is an increasing trend,while the silent or knockdown group is an inhibitory state/moderate4.In the evaluation of cloning ability,the results of the plate cloning assay showed that when compared with the Control group,the cloning ability of the CSE overexpression group was enhanced,and the silence/knockdown group was moderate.5.In the test of cell invasion ability,both soft agar colony formation and invasion showed enhanced invasion ability of CSE overexpression group and weakened/moderate in silencing/knockdown group.6.In the experiment of judging apoptosis,Tunel staining showed that the CSE overexpression group inhibited apoptosis,and the silent/knockdown group promoted apoptosis.7.Cell flow cytometry results show that overexpression of CSE can decrease the S phase of the cell and increase the G2 phase while in silencing/knockdown the results is opposite/opposed.8.Western blotting results showed that after overexpression of CSE,the expression of anti-apoptotic proteins Bcl-2 and Bcl-xl increased,while the expression of pro-apoptotic proteins Bax,Bad,Cleared Caspase3,Cleared Caspase9 and Cleared PARP decreased;invasion-related protein E-cadherin,N-cadherin,MMP-2,MMP-9,TIMP-1,TIMP-2 expression increased,and Vimentin protein expression decreased;cycle-related protein Cyclin D1,Cyclin El,CDK2,CDK4 expression increased,P21,P27 cut back.9.In related pathway studies,the phosphorylation level of AKT/PI3K/mTOR increased after CSE overexpression,while the phosphorylation level of ERK/P38/JNK decreased,while the opposite/opposed results appeared in the silent/knockdown group.So,CSE overexpression promoted cell apoptosis death via the ERK/P38/JNK signaling pathway and regulating cell proliferation is through the AKT/PI3K/mTOR pathway.10.The tumor volume and weight of nude mice injected with cells in the CSE overexpression group were larger than those in the control group,and the results in the silent/knockdown group were reverse/opposite.The positive rates of tumor tissue staining KI67,CD31 and MMP-9 were higher than the control group,while the positive rate of P21 was lower in Control group.ConclusionsCSE,the key enzyme for the endogenous synthesis of hydrogen sulfide,can promote the proliferation,migration,and invasion of human nasopharyngeal carcinoma cells,and regulate its growth through the AKT/PI3K/mTOR and ERK/P38/JNK signaling pathways,thus affecting the occurrence and development of human nasopharyngeal carcinoma cells.