Function And Mechanism Of Succinylation Modification In Gastric Cancer And Clear Cell Renal Cell Carcinoma

Objective: Succinylation modification is a dynamic and reversible post-translational modification(PTM),which can be regulated by a series of enzymes.It has been reported that succinylation modification can regulate the oncogenesis and development of different kinds of tumors.At present,there are four well-known succinylation related enzymes,including two succinyltransferases CPT1 A and KAT2 A,and two desuccinylases SIRT5 and SIRT7.Procollagen-lysine,2-oxoglutarate-5-dioxygenase(PLOD)gene family is one kind of lysine hydroxylases,which contains three members:PLOD1-PLOD3.In previous studies,PLODs were known to participate in the occurrence and development of tumors,including gastric cancer(GC).Our analysis inferred that PLOD2 could act as an independent prognostic factor for both overall survival(OS)and disease-free survival(DFS)of GC patients,and could specifically prompt their risk of peritoneal seeding after surgeon.We firstly found that PLOD2 could be regulated by succinylation modification in GC.In addition,we found that succinylation regulators have unique and important prognostic values in cc RCC.Therefore,based on succinylation modification,this study,on the one hand,aims at revealing the mechanism on how succinylation regulators regulate PLOD2 to promote peritoneal metastasis in GC,on the other hand,clarifying the roles and mechanism of succinylation regulators in cc RCC.Methods: 1.Online datasets were used to analyze the expression of PLOD gene family in GC tissues and adjacent normal tissues,as well as the correlation between the expression of each member and OS or DFS in GC patients.A nomogram prognostic predictive model containing PLOD2 was constructed and the relationship between the expression of PLOD2 and postoperative recurrence or metastasis,especially the peritoneal seeding in patients with GC was also analyzed.2.q RT-PCR and Western blotting were used to verify the expression pattern of PLOD2 among immortalized gastric epithelial cells GES-1 and several gastric cancer cell lines.3.Transwell assay,tumor cell-extracellular matrix adhesion assay and tumor cell-human peritoneal mesothelial cell HMr-SV5 adhesion assay(these three experiments were simply summarized as peritoneal metastasis related experiments as follows)were used to detect the effects of deletion or over-expression of POLD2 on the migration,invasion,adhesion to matrix and HMr-SV5 cells of GC cells in vitro.4.Nude mice were used to establish the peritoneal metastasis model to verify the effect of PLOD2-deletion on peritoneal metastasis of GC in vivo.5.Immunoprecipitation(IP)was used to verify the succinylation modification in PLOD2 as well as the change in succinylation level under the circumstance of adding exogenous succinyl coenzyme A sodium salt or succinic acid disodium salt;6.Peritoneal metastasis related experiments were used to verify the effect of exogenous succinyl coenzyme A sodium salt and succinic acid disodium salt on the ability of peritoneal metastasis in GC cells.7.IP was used to confirm that CPT1 A can bind with PLOD2 so that up-regulate its succinylation level.8.q RT-PCR and Western blotting were used to detect the expression of PLOD2 both at m RNA level and protein level during CPT1A-deletion,respectively.9.Online datasets GSE62254 and TCGA-STAD were used to analyze the correlation between CPT1 A and PLOD2 at m RNA level,respectively.10.Western blotting was used to confirm that MG132 could reverse the inhibition of PLOD2 at protein level by the deletion of CPT1 A combing with Cycloheximide(CHX),and CPT1 A could regulate the expression of PLOD2 through ubiquitin-dependent proteasome pathway.11.Peritoneal metastasis related experiments were performed to confirm that CPT1 A could promote peritoneal metastasis of GC cells via regulating PLOD2;12.The succinylation sites of PLOD2 were predicted by Succinsite and GPSuc online websites,respectively,and the ubiquitination sites of PLOD2 were searched by PLMD online website.13.IP was used to detect the change of succinylation level of PLOD2 under the condition of over-expression of wild-type or site-mutation PLOD2 plasmids.14.Peritoneal metastasis related experiments were used to verify the effect of mutation in K341 on the ability of peritoneal metastasis in GC cells;15.TCGA pan-cancer dataset was used to analyze the expression of the four well-known succinylation regulators in ten common tumors and their impacts on OS of patients,and finally,the unique and important prognostic values in cc RCC were identified.16.Consensus clustering analysis was used to divide cc RCC patients into groups with different survival and clinicopathological characteristics according to the expression similarity of the four succinylation regulators.17.The “Limma” R package was used to identify differently expressed genes(DEGs)among different clusters,and the "cluster profiler" R package was used to perform pathway enrichment using DEGs.18.CIBERSOR website was used to analyze the infiltration of immune cells among different clusters.19.The Immport website,the DEGs as well as the univariate Cox regression analysis of the DEGs were used to find out important immune genes regulated by succinylation regulators,and LASSO Cox was performed to further screen the core immune genes to construct the 6-immune gene-signature.20.Pub Med website was used to search for m6 A methylation regulators,online website PLMD was used to clarify whether there are succinylation sites among m6 A regulators.21.Cc RCC protein dataset from CPTAC website was used to analyze the expression patterns of succinylation regulators and m6 A regulators among cc RCC tissues and normal tissues as well as the relationship between succinylation regulators and m6 A regulators.21.Immunohistochemistry(IHC)was used to detect the expression of four succinylation regulators,LRPPRC and EIF3 B in our own 42 pair of cc RCC samples and para-cancerous tissues,the correlation between CPT1 A and LRPPRC,SIRT5 and EIF3 B,CPT1A and EIF3 B,SIRT5 and LRPPRC were also analyzed.22.Western blotting was used to detect the expression of LRPPRC and EIF3 B in ACHN cells after knockdown of CPT1 A and SIRT5,respectively.Results: 1.PLOD2 could act as an independent prognostic factor for OS and DFS of patients with GC,and it could specifically indicate the risk of peritoneal seeding.Analyses using online datasets showed that all the expression of PLOD1-3 in GC tissues was higher than those in adjacent normal tissues,and PLOD2 could act as an independent prognostic factor for OS and DFS of GC patients,while PLOD1 and PLOD3 had no significantly prognostic value.Based on this,we established a nomogram prognostic predictive model containing PLOD2,and it was verified to have good predictive value.Correlation analysis showed that high expression of PLOD2 could specifically refer the risk of peritoneal seeding in GC patients after surgical operation.2.PLOD2 is highly expressed in GC cells and could promote peritoneal metastasis.Both the expression of PLOD2 at m RNA level and protein level in GC cell lines were higher than those in GES-1.The ability in migration,invasion,adhesion to matrix as well as to HMr-SV5 cells of GC cells was significantly down-regulated during deletion of PLOD2,while over-expression of PLOD2 in BGC823 and KNK7 could up-regulate the ability in peritoneal metastasis of GC cells.3.Up-regulation of succinylation level of PLOD2 could promote the capacity of peritoneal metastasis in GC cells.We firstly used IP experiment to confirm that PLOD2 protein could be regulated by succinylation modification,and then we found that both the succinylation level in GC cells and PLOD2,as well as the ability of peritoneal metastasis in GC cells were up-regulated by adding exogenous succinyl Co A sodium salt and succinate disodium salt.4.CPT1 A could promote peritoneal metastasis of GC cells by regulating PLOD2 through its succinyltransferase activity.In MGC803 cells,PLOD2 was confirmed to bind with CPT1 A by IP;PLOD2 over-expression(OE)plasmid with FLAG-tag and CPT1 A OE plasmid with c-Myc-tag were transfected into in BGC803 GC cells,and then c-Myc tag was confirmed to combine with FLAG-tag.The succinylation level of PLOD2 decreased after silencing CPT1 A in MGC803 cells.5.CPT1 A could regulate the expression of PLOD2 through ubiquitin-dependent proteasome pathway.Only the expression of PLOD2 at protein level rather than at m RNA level was decreased during deleting CPT1 A in MGC803 or BGC823 cell lines.Adding MG132 could reverse the inhibition of PLOD2 by CPT1 A deletion combining with CHX.These results referred that CPT1 A could regulate the expression of PLOD2 through ubiquitin-dependent proteasome pathway.6.K341 is the key site for succinylation modification in PLOD2.After prediction of the succinylation and ubiquitination sites of PLOD2 by online websites,PLOD2-WT,PLOD2-161 R,PLOD2-204 R and PLOD2-341 R OE plasmids with FLAG-tag were transfected into BGC823 cells,respectively.And the succinylation level of PLOD2 with 341 R was significantly lower than that with WT.What’s more,the ability of migration,invasion,adhesion to matrix as well as to HMr-SV5 cells in BGC823 and MKN7 cells transfected by PLOD2-341 R plasmids were significantly up-regulated in comparison to the cells transfected by PLOD2-WT plasmids.7.Pan-cancer analysis suggested that succinylation regulators had unique and vital prognostic values in cc RCC.The results of pan-cancer analysis in ten common tumors(ESCA、STAD、LUSC、LUAD、LIHC、KIRP、cc RCC、COAD、BRCA and BLCA)suggested that only in cc RCC,all the four enzymes have prognostic values for OS of patients.8.Cc RCC patients could be divided into three clusters with significantly different OS and clinicopathological characteristics based on consensus clustering analysis of four succinylation regulators.The patients were divided into three clusters according to the expression similarity of four succinylation regulators.Kaplan Miere survival analysis and correlation analysis between succinylation regulators and clinicopathological parameters showed that there were significantly different OS and clinicopathological characteristics among three clusters.9.Succinylation regulators might promote the malignant progression of cc RCC by regulating the infiltration of immune cells,in which the infiltration of Tregs played an important role.Differential expressed genes(DEGs)analysis and Gene Ontology(GO)pathway enrichment between different clusters showed that a large number of immune related pathways were enriched in clusters with poor prognosis(cluste2 and cluster1).CIBERSORT was used to analyze the infiltration pattern among different clusters and revealed that Tregs,M0 macrophages and resting mast cells were differently infiltrated in three clusters,in which Tregs and resting mast cells could act as a risk factor and protective factor for cc RCC,respectively.Combing with the different infiltration of immune cells,Tregs was inferred to play important role in malignancy of cc RCC.10.Immune signature based on succinylation modification could prominently predict the OS of cc RCC patients.Immune genes under the regulation of succinylation modification were screened by the following principles:(1)the genes were both up-regulated in cluster2 and cluster1(2)the genes have poor prognosis value for cc RCC patients(HR>1,P<0.05);(3)the gene were contained in the immune gene list from Immport website.A total of 34 genes were screened out following above principles.After dimension reduction of Lasso Cox,finally a 6-immune gene signature was constructed.11.Succinylation regulators might promote the malignant progression of cc RCC by regulating m6 A methylation regulators.CPT1 A and SIRT5 could up-regulate and down-regulate LRPPRC and EIF3 B,respectively.Except for immune related pathways,multiple pathways related to RNA regulation were also enriched in cluster2,suggesting that succinylation regulators might regulate the progression of cc RCC by RNA modification,in which the most common pattern is m6 A methylation modification.We used PLMD website to analyze the succinylation modification sites of 32 m6 A regulators and found that there were several succinylation sites in HNRNPA2B1,HNRNPC,HNRNPG(RBMX),LRPRRC and EIF3 B.Analysis using cc RCC protein dataset,results form IHC of our own specimens as well as Western blotting showed that CPT1 A could up-regulate LRPPRC while SIRT5 could down-regulate EIF3 B.Conclusion: 1.Up-regulation of succinylation modification of PLOD2 could promote the ability of peritoneal metastasis in GC cells.2.CPT1 A could up-regulate the succinylation level of PLOD2 via its succinyltransferase activity.3.CPT1 A could regulate the expression of PLOD2 via ubiquitin-dependent proteasome degradation pathway.4.The key succinylation modification site of PLOD2 is K341.5.Succinylation regulators have unique and important prognostic values in cc RCC.6.Succinylation regulators might promote malignant progression of cc RCC by regulating the infiltration of immune cells and the expression of immune related genes,in which the infiltration of Tregs plays an important role.7.Succinylation regulators might promote the malignant progression of ccr RCC by regulating the m6 A regulators,in which CPT1 A and SIRT5 could up-regulate and down-regulate LRPPRC and EIF3 B,respectively.