Study On The Mechanism Of NEK8 Promoting Gastric Cancer Progression By Regulating Asn Metabolism

Background: Gastric cancer(GC)is a malignant tumor with high incidence and mortality,ranking second in the number of new cancer cases and deaths in China.In recent years,with the development of tumor metabolism,studies suggested that amino acid metabolic reprogramming may play an important role in the occurrence and development of GC.However,study on the mechanism of amino acid metabolic reprogramming in GC is still lacking.This study aimed to explore the mechanism of tumor-associated kinase NEK8 promoting GC progression through regulating Asn metabolism.Methods: The expression levels of NEK8,ASNS and ATF4 in GC cells and tissues were detected by immunohistochemistry,RT-PCR and Western Blot.Cell function changes were detected by cell proliferation assay,scratch healing assay,clonogenesis assay,Transwell invasion and migration assay and Ed U proliferation assay.The localization and binding of NEK8 and ASNS in cells were analyzed by immunofluorescence and adjacent ligating techniques,respectively.The changes of cell cycle and apoptosis were detected by flow cytometry.The changes of intracellular molecular metabolism after NEK8 knockdown were detected by untargeted metabolomics.The binding of NEK8 and ASNS in cells and their specific domain were analyzed by immunoprecipitation and plasmid segmentation.The phosphorylation site of ASNS was detected by mass spectrometry,and directed point mutation was used to prevent the phosphorylation of this site to detect whether this site is a functional phosphorylation site.Finally,the effect of phosphorylation site mutation on GC cells in vivo was analyzed by subcutaneous tumorigenesis technique in nude mice.SPSS 22.0software,Graph Pad Prism 7.0 and R Version 3.6.1 were used for statistical analysis of the data involved in this study,and the statistical significance level was set as P=0.05.Results: NEK8 expression was up-regulated in GC and correlated with prognosis of patients with GC.Knockdown or overexpression of NEK8 could inhibit or promote proliferation,clonal formation,cell migration and invasion of gastric cancer cells,respectively.The results of Flow cytometry showed that NEK8 could promote the progression of GC cell cycle and inhibit apoptosis of GC cells.Bioinformatics analysis showed that NEK8 might promote the progression of GC by regulating cellular metabolic pathways.Metabonomics analysis showed that NEK8 could regulate amino acid metabolism of GC cells and also play a role in the m TOCR1 pathway.The results of mass spectrometry analysis and WB showed that NEK8 could regulate the ATF4/ASNS axis,and then regulate Asn metabolism.As an intermediate molecule of NEK8 regulating Asn metabolism,ATF4 can also promote the proliferation,invasion and migration of GC cells.Moreover,we also constructed an accurate nomogram to predict the prognosis of GC based on ATF4(C index = 0.797,AUC = 0.855 for 3-year survival rate and AUC = 0.863 for 5-year survival rate).Mechanistically,NEK8 phosphorylates ASNS at S349,which inhibits the proliferation of GC in vivo and in vitro.Finally,the inhibitory effect of NEK8 knockdown on m TOCR1 pathway can be compensated by exogenous Asn supplementation,suggesting that NEK8 regulates m TOCR1 pathway through Asn.Conclusion: NEK8 is overexpressed in GC tissues and cells,and can promote the proliferation,invasion,migration and clonal formation of GC cells.Mechanically,NEK8 can regulate Asn metabolism by regulating ATF4/ASNS axis and S349 phosphorylation of ASNS,and then regulate m TORC1 pathway,ultimately leading to the GC progression.