Mechanism Of TET2 Inhibiting The Progression Of Nasopharyngeal Carcinoma Through Glycolytic Pathway

Objective: According to the data published by the International Center for Research on Cancer(IARC)in 2020,nasopharyngeal carcinoma(NPC)ranks among the top 30 in the number of new tumors and deaths in the world,and it is also one of the most common malignant tumors in my country.In Southeast Asia and North Africa,my country is more common in Guangdong,Guangxi and other places,is the third most common cancer in southern my country.The occurrence of nasopharyngeal carcinoma is related to EB virus infection,environmental factors,genetic factors and other factors,but the pathogenesis is still unclear.Due to the occult location and high invasiveness of nasopharyngeal carcinoma,80% of clinical patients are in the middle and late stages when they are first diagnosed.Radiation therapy is currently the main treatment for nasopharyngeal carcinoma.After radiotherapy and concurrent adjuvant chemotherapy,the clinical outcome of nasopharyngeal carcinoma patients has improved,but tumor recurrence and distant metastasis are still difficult problems,and the incidence rate is 20-30%.TET(ten-eleven translocation)protein family,including TET1,TET2 and TET3,is a dioxygenase present in organisms that catalyzes the conversion of 5-methylcytosine(5-m C)to 5-hydroxymethylcytosine(5-hm C)demethylates DNA to regulate gene transcription.TET protein is a star protein in the field of epigenetics in recent years.5-m C and 5-hm C are essential epigenetic proteins in almost all metabolic pathways of cells.genetic markers.TET proteins play a role in the development of a variety of diseases,and many studies have pointed out that TET2 mutations are associated with a variety of hematological diseases,such as acute myeloid leukemia,chronic myelomonocytic leukemia,angioimmunoblastic T-cell lymphoma,and lung cancer,liver cancer,breast cancer and other malignant tumors are related to the occurrence and drug resistance.However,the role of TET2 in tumors is diverse.In some tumors,it plays a role in inhibiting tumor development,but in other tumors,its role is just the opposite.Overactive aerobic glycolysis is one of the important markers of tumor metabolism and is crucial for maintaining tumor growth.Its main effects on tumor cells are increased glucose uptake and increased lactate secretion.The glycolysis process is strictly regulated by a variety of enzymes,and the main reason for the enhancement of the glycolysis capacity of tumor cells is also related to the expression and activity of key glycolysis enzymes.Most of the enzymes involved in glycolysis(hexokinase),phosphofructokinase,pyruvate kinase,lactate dehydrogenase)have been reported to be overexpressed in various tumors,such as lung cancer,liver cancer and nasopharyngeal carcinoma.In this experiment,we first confirmed the effect of TET2 on the biological function of nasopharyngeal carcinoma cells through in vivo and in vitro experiments,and carried out mechanism research from the perspective of protein-protein interaction to determine the TET2-interacting protein pyruvate kinase PKM,and analyze TET2 The site and fine regulation of the interaction with PKM,to explore the effect of TET2 and PKM on aerobic glycolysis in nasopharyngeal cancer cells,and hope to provide new targets for the treatment of nasopharyngeal cancer in the future.Methods: The main subjects of this study were human nasopharyngeal cells NP69,nasopharyngeal carcinoma cells CNE1,HNE1,5-8F and SUNE-1,and 120 4-week-old male BALB/c nude mice.1.Determine the research protein TET2: cell culture,RNA extraction and q RT-PCR,etc.2.To explore the effect of TET2 on the biological function of nasopharyngeal carcinoma cells: To study the effect of TET2 on the proliferation and invasion of nasopharyngeal carcinoma cells by plasmid transfection,CRISPR/cas9,CCK8,Transwell,and subcutaneous transplanted tumors in nude mice.3.Co-immunoprecipitation combined with mass spectrometry analysis to identify TET2 interacting protein PKM: mass spectrometry analysis,Co-IP,Western blot,Dot-blot,etc.4.To explore the role of TET2 and PKM and its fine regulation: Co-IP,Western blot,CHIP,extraction of cytoplasm and nucleus,etc.5.Detect the effects of TET2 and PKM changes on glycolysis in nasopharyngeal carcinoma cells: cell transfection,tumorigenic experiment in nude mice,glucose uptake,lactate release,phosphoenolpyruvate production,p H value detection,etc.Results: 1.The expression abundance of TET2 in each nasopharyngeal carcinoma cell line is higher than that of TET1 and TET3,so TET2 was selected as the main research object of this experiment.2.The proliferation and invasive ability of nasopharyngeal cancer cells were weakened after overexpression of TET2,and the proliferation and invasive ability of nasopharyngeal cancer cells were enhanced after knockdown and knockout of TET2.3.The ability of TET2 to inhibit the proliferation and invasion of nasopharyngeal carcinoma cells does not depend on its catalytic activity.4.PKM is the interacting protein of TET2.The N-terminus of TET2 interacts with PKM in the cytoplasm,thereby inhibiting the proliferation and invasion of nasopharyngeal carcinoma cells.5.TET2 had no significant effect on the pre-transcriptional,transcriptional and protein levels of PKM.6.After knocking out TET2,the glucose uptake of nasopharyngeal carcinoma cells was increased,the p H value was decreased,the production of lactate and PEP was increased,and the glycolysis was enhanced.Further knockdown of PKM significantly reversed the effects of TET2 knockdown on glucose uptake and p H.7.Adding the PKM inhibitor shikonin can inhibit the production of lactate and PEP induced by TET2 knockout,and inhibit the proliferation and invasion of nasopharyngeal carcinoma cells.8.To establish a mouse xenograft model,the tumor volume of the TET2 knockout group increased,and the survival rate was lower than that of the control group.After intravenous injection of shikonin,the tumor volume of the TET2 knockout group was reduced,and the survival rate was improved.Conclusions: 1.TET2 can inhibit the proliferation and invasion of nasopharyngeal carcinoma cells.2.PKM is an interacting protein of TET2.3.The N-terminus of TET2 interacts with PKM in the cytoplasm.4.TET2 interacts with PKM to inhibit the progression of nasopharyngeal carcinoma through the glycolysis pathway.