The Role And Mechanism Of DTYMK In Malignant Progression Of Lung Cancer

Objective: In the world,the death rate of lung cancer ranks the first among malignant tumors,and it has become the most dangerous cancer to human health.Identification of new markers and therapeutic targets for lung cancer is of great significance for early detection,diagnosis and treatment of lung cancer.Deoxythymidine kinase DTYMK is a key enzyme involved in the biosynthesis of d TDP,which plays an important role in the synthesis of d TTP and the maintenance of genomic stability.At present,there are few studies on DTYMK in tumor.This study aims to clarify the expression level of DTYMK in lung cancer and its clinical significance,and reveal the role and regulatory mechanism of DTYMK in the development of lung cancer,so as to provide experimental evidence for the early diagnosis of lung cancer and new target for tumor treatment.Methods:(1)Bioinformatics method was used to analyze the expression level and clinical significance of DTYMK in pan-cancer,especially in lung cancer.(2)Si RNA transfection silenced the expression level of DTYMK gene in lung cancer cells.(3)CRISPR/Cas9 technique was used to construct stable DTYMK knockdown cell lines.(4)The growth and proliferation ability of lung cancer cells were detected by cell growth curves,plate colony formation assays and Ed U assays in vitro.(5)The effect of DTYMK on cell migration was determined by cell scratch assay.(6)Cell cycle was detected by flow cytometry.(7)The extracellular DTYMK content was determined by ELISA.(8)The in vivo antitumor effect of inhibition of DTYMK was investigated by the xenograft mouse models.(9)The proteins interacting with DTYMK were identified by mass spectrometry and verified by immunocoprecipitation.(10)RNA sequencing.(11)Ch IP-q PCR explored upstream transcription factors that regulate DTYMK.(12)Determination of mitochondrial DNA and cell oxygen consumption rate.Results:(1)Bioinformatics analysis showed that pyrimidine anabolism was up-regulated in a variety of cancer cells,while DTYMK,as a key enzyme in pyrimidine anabolism,was up-regulated in a variety of cancer cells.(2)Bioinformatics analysis showed that DTYMK expression affected the tumor immune microenvironment.(3)Bioinformatics analysis showed that the expression of DTYMK in tumor cells was significantly increased in the single cell expression profile.(4)Bioinformatics analysis showed that in various types of tumors,the expression level of DTYMK could be distinguished between tumor tissues and normal tissues,and high expression was associated with poor prognosis of patients.(5)Bioinformatics analysis and experimental results showed that DTYMK was highly expressed in lung cancer cells and associated with poor prognosis.(6)DTYMK protein could be detected,both in the cell and in the supernatant,to distinguish lung cancer cells from normal cells.(7)Silencing or stable knockdown of DTYMK inhibited the growth and proliferation of lung cancer cells.(8)Silencing or stable knockdown of DTYMK caused the stagnation of lung cancer cells in the S phase and weakened the migration ability of lung cancer cells.(9)NRF1 directly regulates the transcription of DTYMK gene.(10)Inhibition of DTYMK reduced mt DNA and oxygen consumption of lung cancer cells.(11)DTYMK interacts with PHB1,and inhibition of DTYMK significantly down-regulates the expression of PHB1 protein.(12)Exogenous overexpression of PHB1 can partially restore the reduction of mt DNA and oxygen consumption in lung cancer cells after targeted inhibition of DTYMK.Conclusions: The pyrimidine synthesis flux was significantly increased in tumor cells,and DTYMK,as a key enzyme regulating the pyrimidine synthesis flux,especially thymine synthesis flux,was significantly increased in tumor cells.DTYMK has important clinical significance.It can not only affect the immune microenvironment,but also has diagnostic and prognostic value for a variety of tumors.DTYMK can affect various biological behaviors of lung cancer cells and contribute to the occurrence and development of lung cancer.DTYMK is mainly located in mitochondria,which is directly regulated by transcription factor NRF1,and can significantly affect mitochondrial function through its interaction with mitochondrial protein PHB1.This suggests that DTYMK is a novel tumor therapy target that can combine targeting the pyrimidine synthesis pathway and mitochondria.