Study On The Role And The Related Mechanism Of PGK1 And Its Lysine Succinylation Modification In Renal Cell Carcinoma

Renal cell carcinoma(RCC)is the most common type of kidney malignancy renal cancer in adults.The epidemiological data has showed that,the incidence of RCC has been increasing year by year.Due to the complex characteristics of the disease and the diversity of genetics and histology,RCC is lacking in appropriate clinical specific molecular diagnostic markers.Therefore,there is still lack of effective means for the early diagnosis,prognosis evaluation,postoperative tracking and the evaluation of target treatment effect of RCC.Recent years,studies on the pathogenesis and development of RCC have shown that the inactivation of VHL mutation(a tumor suppressor gene)and its downstream HIF-VEGF(hypoxia inducible factor)pathway activation based hypoxia induced neovascularization is one of the most important molecular pathological of RCC.Meanwhile,metabonomics study of RCC suggests that under hypoxia,the remodeling of energy metabolism network may be an important part of RCC development.Therefore,the regulation of tumor metabolism has become a hot topic in the treatment of renal cancer in recent years.Posttranslational modifications refers to the process of protein covalent experience in translation,usually through the process of its specific amino acid site residues plus modified groups or proteolytically cut groups.By regulating the activity of the protein,stability,subcellular localization and protein interactions,promote the function of the protein diversity,PTMs is an effective mechanism for expanding gene encoding,regulate the physiological functions of cells,and thought to be related with almost all known cellular pathways in diseases.With the development of mass spectrometry,more and more PTMs types have been identified.Researches have shown that PTMs plays a crucial role in different biological processes,such as cell regulation and differentiation,organism development and multiple substance metabolism.Abnormal PTMs of proteins may be closely related to multiple diseases,including tumors.Lysine succinylation modification is a newly discovered PTMs,and it is thought to be a process of covalently binding the succinyl group to the lysine residue by enzymology or non enzymology.Succinylation is a process in which a negatively charged four carbon succinyl group is covalently combined with a primary amine of lysine residue by enzymology or non enzymology.Compared with the modification of methylation and acetylation,lysine succinylation can transfer larger structural groups and induce a greater change in the charge of protein.Therefore,the protein lysine succinylation may promote more obviously substantial alternations in the structure and function of protein.At present,identification and analysis by mass spectrometry indicate that many metabolic enzymes,including glycolysis,three carboxylic acid cycle and key enzymes of fatty acid metabolism,have succinylated modification.This may suggest that lysine succinylation is widely involved in the biological processes associated with energy metabolism and plays an important role in regulation and control.However,studies on the mechanism of protein lysine succinylation is still not clear.There are few reports on the role of lysine succinylation modification on tumor progression.Therefore,this study focus on the identification and analysis of the quantitative global proteome and lysine succinylation of RCC to investigate its potential effects of the global proteome and lysine succinylation alternations on energy metabolism of RCC,and the results of this study can provide new ideas and direction for the diagnosis and treat target of RCC.Methods1.Sample collection1.1 Collection of RCC samplesIn this study,the collection of renal cell carcinoma samples was approved and supported by the ethics committee of the First Affiliated Hospital of China Medical University.We collected 9 pairs of renal cell carcinoma and its corresponding adjacent tissues for identification and analysis of LC-MS/MS.We also collected 10 pairs of RCC and the adjacent tissue liquid nitrogen frozen specimens for future analysis,and 45 pairs of RCC and the adjacent paraffin embedded specimens for immunohistochemical staining.These specimens were taken from ho the patients who underwent surgical treatment of RCC during September 2015 to December 2017 at the Department of Urology,the First Affiliated Hospital of China Medical University,w.All patients signed the informed consent of scientific research before operation.All the patients were not treated with any treatment before operation.All the specimens were diagnosed as clear cell carcinoma of the kidney.1.2 Cell cultureIn this study,the OSRC,769-P and ACHN cell lines were used for the followed tests.The cell lines were from the Department of Urology,the First Affiliated Hospital of China Medical University.2.Analysis process2.1 Quantitative analysis of proteome and lysine succinylation modification in RCCLC-MS/MS was used to identify proteome expressions and quantify the differential expression protein in RCC and the adjacent tissues.Enrichment of succinylated proteins by the use of succinylated pan-Ksucc antibody,and the LC-MS/MS was used to identify the expression of succinylated modifications of proteins group,and quantified differential expression sites and related proteins.2.2 Enrichment and functional analysis of differentially expressed proteins and differentially expressed succinylated modification proteinsGO and KEGG pathway were used to enrich the identified differentially expressed proteins and the succinylated modification sites,and clarify the biological processes that may be involved in identifying differential proteins,especially differently succinylated modifications.2.3 Analysis of the correlation between the differentially expressed proteins and the differentially expressed succinylated modification sites of these proteinsWe constructed a protein-protein interaction network,standardized the different succinylation sites,identified the correlation between differentially expressed proteins and differentially expressed proteins succinylation sites,and screened potential potential targets.2.4 Detection of the expressions of PGK1 and PKM2 in RCCThe expressions of gene levels of PGK1 and PKM2 were extracted and analyzed in Oncomine database and TCGA database.Expressions of PGK1 and PKM2 protein in RCC were detected by Western blot.2.5 Analysis of the clinical significance of PGK1 proteinImmunohistochemical detection of the in situ expression of PGK1 in RCC and analyze the correlation between the expressions of PGK1 and the histopathological classification of RCC.2.6 Detection of succinylated modification of PGK1 RCCPGK1 protein was enriched by immunoprecipitation in RCC and adjacent tissue,and pan-Ksucc antibody was used to detected the PGK1 succinylation.2.7 Detection of the PGK1's effect on biological behavior in RCC cell linesSi-RNA technology was used to interfere the endogenous expression of PGK1 in RCC cell lines OSRC and ACHN.The proliferation ability of cells was detected by CCK8 cell proliferation assay,and the migration ability of cells was detected by Transwell assay.2.8 Detection of SIRT5's effect on PGK1 succinylation modificationThe expression of SIRT5 gene in RCC was extracted by Oncomine database.SIRT5 was overexpressed in ACHN cell line of RCC,PGK1 was enriched by immunoprecipitation,and the levels of PGK1 succinylated expression was detected by Western blot.2.9 Detection of the SIRT 5's effect on biological behavior in RCC cell linesThe proliferation ability of cells was detected by CCK8 cell proliferation assay,and the migration ability of cells was detected by Transwell assay.2.10 Statistical analysisThe experimental data were all displayed with mean standard deviation(Mean ± SD),and the statistical analysis was carried out by SPSS 16 software.Paired sample t test was used for paired sample data,and independent sample t test was used among random sample groups.The standard setting of statistical difference was P < 0.05.Results1.Quantitative analysis of differentially expressed proteins and differentially expressed succinylated modification in RCCThrough LC-MS/MS analysis,we identified 4116 proteins from these 9 RCC tissue samples,of which 3217 proteins could be quantified.After threshold screening,668 proteins were expressed differently in tumor tissues and adjacent tissues,including 189 up-regulated and 479 down regulated proteins.Of these 9 pairs of RCC tissue samples,we identified 1668 lysine succinylated(Ksucc)sites in 1090 proteins,of which 1238 Ksucc sites in 844 proteins were quantified.Compared with adjacent tissue,we detected regulated 51 Ksucc sites in 47 proteins and up dowen-regulated 110 Ksucc loci in 95 proteins.2.Enrichment and functional analysis of differentially expressed proteins and differentially expressed succinylated modification proteinsBy the GO enrichment and KEGG pathway analysis of differentially expressed proteome and differentially expressed succinylated modifications,we found that the up regulated succinylated proteins were enriched in glycolysis,amino acid biosynthesis and hHIF-1 signaling pathway related proteins.In contrast,down-regulated succinylated proteins were enriched in the pathway of carbon metabolism,TCA cycle and fatty acid metabolism.We further analyzed the associations of succinylation sites and their corresponding protein expressions.Succinylated modification of HspB1,FGA,COL6A3,FN,PGK1,PKM and some other proteins was the most significant.It needs to be further verified at the level of tissue and cell.3.The Expression and significance of PKM2,PGK1 and the succinylated modification in RCCWe extracted the chip data of PGK1 and PKM2 genes in cancer by Oncomine database.We found that PGK1 and PKM2 mRNA showed high expressions in RCC tissues.The expressions of PGK1 and PKM2 protein in the tissues were of the same high in the Western blot analysis.After immunoprecipitation and enrichment of PGK1 and PKM2 proteins,we found that the succinylation levels of these two proteins were also high.Immunohistochemistry results also showed that PGK1 was high expression in RCC tumor tissues and located in cytoplasm and nucleus.There was a significant positive correlation between the staining score and the pathological grade of RCC.4.Detection of PGK1 on proliferation and migration of RCC cell linesWe constructed PGK1 specific siRNA by RNAi technique,which inhibited the level of PGK1 expression in the RCC cell line OSRC and ACHN.The results of CCK8 cell proliferation test showed that after transfection of siRNA,the cell proliferation ability of OSRC and ACHN decreased significantly compared with the control group.The results of Transwell cell migration test showed that after transfection of siRNA,the cell migration ability of OSRC and ACHN was significantly inhibited compared with the control group.5.Detection of SIRT5 on the succinylation of PGK1 and its effect on the proliferation and migration of RCC cellsWe first analyzed the gene chip data of RCC tissue in the Oncomine database,and found that SIRT5 m RNA was significantly reduced in renal cell carcinoma tissue.We constructed SIRT5 over-expression plasmids in ACHN cells to increase the endogenous expression of SIRT5 in the cells.The enrichment of PGK1 by immunoprecipitation and the detection of antibody with pan-Ksucc antibody showed that the expression of PGK1 succinylations in SIRT5 ACHN cell lines was significantly inhibited.The results of CCK8 and Transwell showed that the proliferation and migration of RCC cells decreased significantly after overe-xpression of SIRT5.Conclusion1.Lysine succinylation modification is high abundance PTMs in RCC,and differentially expressed in glycolysis,oxidative phosphorylation,TCA cycle,fatty acid metabolism and energy metabolism compared with the adjacent tissues.2.The key enzymes in glycolysis PKM2,PGK1 and the lysine succinylation of these enzyms are highly expressed in RCC.The results of immunohistochemistry showed that PGK1 showed high expression in RCC tumor tissue and located in cytoplasm and nucleus.There was a significant positive correlation between the staining score and the pathological grade of RCC.3.PGK1 can significantly affect the proliferation and migration of RCC cell lines.4.SIRT5 is low expressed in RCC,and it may regulate succinylation modification of PGK1 through its succinylase activity,thereby affecting the proliferation and migration ability of RCC.