| Objective: Tumor metabolic reprogramming(Energy metabolism reprogramming)is one of the characteristics of tumors,and it is also a hot spot of tumor research in recent years.Metabolic pathways are highly conserved across species,while metabolic enzymes change dynamically in complex ways.Changes in genes encoding metabolic enzymes are one of the important mechanisms of metabolic reprogramming in tumors.In tumor cells,the genes of metabolic enzymes are often mutated or amplified,which in turn changes the levels of metabolites and accelerates the proliferation of cancer cells through their "classical" enzymatic functions.In addition,a variety of metabolic enzymes also exhibit diverse "non-classical" functions.Identifying the "classical" and "non-classical" functions of metabolic enzymes in tumors will help us link complex metabolic phenotypes with intuitive molecular phenotypes for early tumor screening and biomarker screening.Gastric cancer(GC)is the fifth most prevalent tumor in the world,with poor prognosis and strong heterogeneity.Although relevant metabolic studies in the field of gastric cancer have been carried out,the results of different studies are poorly consistent.At present,the research on the metabolic changes of GC is relatively insufficient,and the role and mechanism of metabolic enzymes in the progression of gastric cancer have not been thoroughly studied.In this study,we used metabolomics to screen out the significantly activated metabolic pathway-sphingolipid synthesis pathway in gastric cancer.We explored the role of Serine Palmitoyltransferase Long Chain Base Subunit 1(SPTLC1),a key enzyme in this pathway,in gastric cancer.On the one hand,we confirmed the metabolism-dependent "canonical" function of SPTLC1 in gastric cancer,on the other hand,we also discovered the "non-canonical" function of silencing SPTLC1 to induce mitotic catastrophe(MC)in gastric cancer cells and its mechanism.This study provides a new idea for the diagnosis and treatment of gastric cancer.Methods: 1.Based on the UPLC-MS/MS detection platform,the whole-spectrum metabolome was detected in the cancer tissues and adjacent tissues from 8 gastric cancer patients,and the software Analyst 1.6.3 was used to process the mass spectrometry data for metabolite qualitative and quantitative analysis.Screening of differential metabolites using univariate statistical analysis and multivariate statistical analysis methods;2.Using online datasets to analyze the expression of SPT subunits SPTLC1 and SPTLC2 in gastric cancer tissues and adjacent tissues,and compare the expression differences of SPTLC1 and SPTLC2 in gastric cancer tissues;3.Use si RNA to transfect cells to knock down the target gene,and use the FLAG-tagged overexpression plasmid to achieve SPTLC1 overexpression;4.The expression and knockdown and overexpression efficiency of SPTLC1 and SPTLC2 in gastric cancer cell lines were verified by q RT-PCR and Western blotting experiments;5.The effect of knockdown of SPTLC1,SPTLC2 and plasmid overexpression of SPTLC1 on gastric cancer cell proliferation was verified by cell proliferation inhibition assay(MTS method)and colony formation assay;6.Build a nude mouse subcutaneous tumorigenesis + in vivo si RNA intratumoral injection model for in vivo experiments to verify the changes in gastric cancer cell proliferation after knockdown of SPTLC1;7.Detect the expressions of SPTLC1 and Ki-67 in subcutaneous tumors of nude mice by immunohistochemistry;8.Use sphingomyelin kit to detect the changes of sphingomyelin after knockdown of SPTLC1 in gastric cancer cells;9.Based on the UPLC-MS/MS detection platform,extensive targeted lipid metabolomic analysis was performed to detect the changes in lipid levels after knockdown of SPTLC1 in SNU-216 cells;10.Cell proliferation inhibition assay(MTS method)was used.The reversion experiment was carried out to verify the changes in the proliferation ability of gastric cancer cells after exogenous supplementation of sphingolipid metabolites after knockdown of SPTLC1;11.Enrichment analysis was performed using the Hallmark data set and KEGG data set in GSEA to predict the related pathways of high expression of SPTLC1 in gastric cancer 12.Immunofluorescence(IF)was used to detect the colocalization of SPTLC1 and spindle in cells;13.Flow cytometry was used to detect the cell cycle changes of gastric cancer cells after knockdown of SPTLC1;14.Immunofluorescence was used to detect Gastric cancer cells suffered from mitotic catastrophe after knockdown of SPTLC1;15.Co-immunoprecipitation(co-IP)combined with mass spectrometry were used to screen SPTLC1 interacting proteins;16.Verifying SPTLC1 interacting proteins in different cells using single-cell datasets17.The co-localization of KIF4 A and PRC1 in gastric cancer cells was detected by immunofluorescence;18.The binding of SPTLC1 to KIF4 A and PRC1,and the binding of KIF4 A to PRC1 were confirmed by co-IP;19.The knockdown of gastric cancer cells was confirmed by co-IP The binding of KIF4 A to PRC1 decreased after SPTLC1 was subtracted;20.Zdock software was used for protein-protein molecular docking,and Pymol2.3.0 was used for interaction pattern analysis;21.Co-IP was used to verify the binding site of SPTLC1 and KIF4A/PRC1;22.The cell proliferation ability of knockdown/overexpressing SPTLC1 gastric cancer cells treated with paclitaxel was detected by MTS assay.Results: 1.The results of metabolomic analysis based on LC-MS/MS indicated that sphingolipid metabolites in human gastric cancer tissues were elevated.The results of metabolomic difference analysis between cancer tissues and adjacent tissues of 8 gastric cancer patients showed that some sphingolipids were significantly upregulated in gastric cancer tissues compared with adjacent tissues.2.Online data and cell experiments confirmed that SPTLC1 was highly expressed in gastric cancer.(1)Three gastric cancer data sets such as TCGA were used to compare the expression differences of SPTLC1 and SPTLC2 in gastric cancer tissue and adjacent tissue.SPTLC1 was highly expressed in cancer tissue in all three data sets,while SPTLC2 was only indicated in TCGA data set.The expression of SPTLC1 in gastric cancer cells was higher than that of SPTLC2 by TCGA analysis.(2)The expression of SPTLC1 and SPTLC2 in gastric cancer cell lines was compared by q RT-PCR experiment and Western blotting experiment.Whether it was m RNA level or protein level,SPTLC1 The expression of SPTLC1 was significantly higher than that of SPTLC2.3.In vitro experiments confirmed that SPTLC1 promoted the proliferation of gastric cancer cells while SPTLC2 had no significant effect on the proliferation of gastric cancer cells.The results of cell proliferation inhibition assay(MTS method)and colony formation assay indicated that knockdown of SPTLC1 in SNU-216 and SGC-7901 cells reduced cell proliferation,while overexpression of SPTLC1 enhanced cell proliferation.However,there was no significant change in cell proliferation after knockdown of STPLC2 in these two cell lines.4.In vivo experiments confirmed that silencing SPTLC1 could inhibit the proliferation of gastric cancer cells.The subcutaneous tumorigenesis model of nude mice was constructed for in vivo experiments,and it was found that the growth rate of subcutaneous tumors in mice was slowed down after knockdown of SPTLC1.Immunohistochemistry of tumor sections showed that SPTLC1 and Ki-67 were decreased in si SPTLC1 group.5.Silencing SPTLC1 reduced sphingolipid metabolites in gastric cancer cells,and exogenous supplementation of sphingolipid metabolites only partially restored the proliferation inhibition caused by silencing SPTLC1.(1)The detection results of sphingomyelin kit indicated that sphingomyelin decreased after knockdown of SPTLC1 in SNU-216 and SGC-7901cells;(2)The results of targeted lipid metabolomics indicated that sphingolipids were knocked down after knockdown of SPTLC1 in SNU-216 cells(3)The results of recovery experiments showed that exogenous supplementation of sphingolipid metabolite sphingosine-1-phosphate after knockdown of SPTLC1 could only partially reverse the proliferation inhibition caused by knockdown of SPTLC1;6.SPTLC1 regulates the mitotic process of gastric cancer cells.(1)The results of GSEA enrichment analysis indicated that the high expression of SPTLC1 in gastric cancer was enriched in G2 M checkpoint,mitotic spindle,cell cycle and other mitosis-related pathways;(2)The results of immunofluorescence experiments indicated that SPTLC1co-localized with the spindle in SNU-216 and SGC-7901 cells;(3)The results of flow cytometry indicated that SNU-216 and SGC-7901 cells were arrested in the G2/M phase of gastric cancer cell cycle after knockdown of SPTLC1;(4)Immunofluorescence experiments suggest that gastric cancer cells undergo mitotic catastrophe after knockdown of SPTLC1 in SNU-216 and SGC-7901 cells.7.Silencing SPTLC1 in gastric cancer cells reduces the binding of KIF4 A to PRC1 and causes mitotic catastrophe.(1)Co-immunoprecipitation(co-IP)combined with mass spectrometry analysis results to screen the interacting proteins of SPTLC1,and used the single-cell data set to analyze,the results suggest that the expression of SPTLC1 interacting proteins is higher in epithelial cells,And the expression is higher in G2/M phase cells.(2)The results of immunofluorescence experiments indicated that KIF4 A co-localized with PRC1 in SNU-216 and SGC-7901 cells;(3)The co-IP experiment confirmed that SPTLC1 binds to KIF4 A and PRC1,and that KIF4 A and PRC1 bind to each other;the binding of KIF4 A to PRC1 decreases after SPTLC1 knockdown in SNU-216 and SGC-7901 cells;(4)Molecular docking using ZDOCK software indicated that both the N-terminal domain and C-terminal domain of SPTLC1 could bind to KIF4 A and PRC1,respectively;(5)The co-IP results suggest that KIF4 A binds to the N-terminal domain of SPTLC1,and that PRC1 binds to both the N-terminal and C-terminal domains of SPTLC1;8.The high expression of SPTLC1 has a potential role in reducing the sensitivity of gastric cancer cells to paclitaxel.The results of the cell proliferation inhibition experiment showed that compared with the SGC-7901 cells that did not interfere with the expression of SPTLC1,the cells with SPTLC1 knockdown had a lower survival rate after a certain concentration of paclitaxel,and the overexpression SPTLC1 group had a higher cell survival rate after a certain concentration of paclitaxel.Conclusion: 1.The SPT subunit,the key enzyme of sphingolipid synthesis,is highly expressed in gastric cancer,and the sphingolipid synthesis pathway is activated in gastric cancer;SPTLC1 is a more important subunit in promoting the proliferation of gastric cancer cells,and its high expression promotes cell proliferation and sphingolipid synthesis.Synthetic,but SPTLC1 pro-proliferation is ’partially metabolism-dependent’.2.SPTLC1 is a key protein in the mitotic process of gastric cancer cells,silencing SPTLC1 inhibits the formation of KIF4A/PRC1 complex,and then induces mitotic catastrophe in gastric cancer cells;3.The high expression of SPTLC1 has a potential role in reducing the sensitivity of gastric cancer cells to paclitaxel. |
PDF Full Text Request