The Role And Mechanism Of TKT Succinylation In Platelet Promotion Of Intrahepatic Cholangiocarcinoma Proliferation And Metastasis

Objective: Intrahepatic cholangiocarcinoma is a highly lethal hepatobiliary tumor,the incidence of which is increasing every year.Accumulating evidence suggests that crosstalk between platelets and cancer cells promotes tumor proliferation and metastasis.Therefore,the aim of this study is to investigate the role and mechanism of the interaction between platelets and intrahepatic cholangiocarcinoma,which in turn promotes the progression of intrahepatic cholangiocarcinoma,and to provide new strategies for the treatment of intrahepatic cholangiocarcinoma.Methods: To investigate the correlation between platelet-related indicators and clinicopathological features of intrahepatic cholangiocarcinoma,the preoperative platelet count,the degree of platelet activation and the extent of platelet infiltration in the microenvironment of patients with intrahepatic cholangiocarcinoma were explored.Platelets were extracted and isolated from peripheral blood of healthy adults and cocultured with intrahepatic cholangiocarcinoma cells.The effect of platelet induction of EMT in intrahepatic cholangiocarcinoma cells was assessed using Western blotting(WB),immunofluorescence(IF)and q PCR.The effects of platelets promoting proliferation and metastasis of intrahepatic cholangiocarcinoma cells were investigated using the Ed U assay and Transwell invasion and migration assay,and the results of the cellular experiments were validated by subcutaneous transplantation tumor and lung metastasis assays in nude mice.Succinylation modification histology was used to identify key modification proteins and sites for succinylation modification of intrahepatic cholangiocarcinoma cells after incubation with platelets.The TKT K319 site mutation plasmids TKT-K319 E and TKTK319 R were constructed and ubiquitination assays were used to investigate the mechanism of succinylation modification in regulating TKT protein stability.Western blot and immunoprecipitation(IP)assays were used to screen for succinyltransferases and desuccinylases that regulate the succinylation modification of TKT.The effects of TKT and TKT-K319 mutants on the biological behaviour of intrahepatic cholangiocarcinoma cells were evaluated by Ed U assay,CCK8 assay,Transwell invasion and metastasis assay and animal experiments.Immunohistochemical(IHC)analysis was used to investigate the relationship between TKT-K319 hypersuccinylation modification and clinicopathological characteristics and prognosis of patients with intrahepatic cholangiocarcinoma.Healthy human platelets were extracted and incubated with intrahepatic cholangiocarcinoma cells.Flow cytometry and immunofluorescence were used to detect the effect of TKT-K319 hypersuccinylation modification on platelet activation and aggregation in cholangiocarcinoma cells.Result: Preoperative platelet count correlates with prognosis in patients with intrahepatic cholangiocarcinoma;Platelet aggregation in the microenvironment of intrahepatic cholangiocarcinoma and correlates with prognosis;Increased platelet activation in patients with intrahepatic cholangiocarcinoma compared to healthy subjects.After co-incubation with intrahepatic cholangiocarcinoma cells,platelets promoted epithelial-mesenchymal transition(EMT),proliferation and metastasis of intrahepatic cholangiocarcinoma cells.And platelets were able to increase the level of succinylation modification of intrahepatic cholangiocarcinoma cells.Mass spectrometry identified the key modifier protein,TKT,and the modifier site,K319.Intrahepatic cholangiocarcinoma cells with high expression of TKT showed higher proliferation and invasive metastatic ability,and knockdown of TKT inhibited proliferation and invasion of cholangiocarcinoma cells.Immunoprecipitation assays revealed that platelets promote the succinylation of TKT-K319 in cholangiocarcinoma cells and that mock-succinylated TKT-K319 E could inhibit its own deubiquitination by removing the K48 ubiquitin chain.In addition,CPT1 A and SIRT5 act as TKT succinyltransferase and desuccinylase,respectively.In vitro and in vivo experiments showed that the highly succinylated modification of TKT-K319 enhanced the proliferation and metastasis of intrahepatic cholangiocarcinoma cells.In pathological tissues of intrahepatic cholangiocarcinoma,higher levels of TKT-K319 succinylation modification were associated with the prognosis of intrahepatic cholangiocarcinoma patients and may be a prognostic risk factor.Intrahepatic cholangiocarcinoma cells with high levels of TKT-K319 succinylation modification were able to promote platelet activation and aggregation after co-incubation with platelets.Conclusions: Preoperative platelet count correlates with prognosis in patients with intrahepatic cholangiocarcinoma;higher platelet aggregation in the microenvironment of patients with intrahepatic cholangiocarcinoma correlates with prognosis;higher platelet activation in patients with intrahepatic cholangiocarcinoma.Incubation of platelets with intrahepatic cholangiocarcinoma cells promoted the succinylation modification of TKTK319 and stabilised its own expression by removing the K48 ubiquitin chain,thereby enhancing the proliferation and metastasis of intrahepatic cholangiocarcinoma cells.The high level of TKT-K319 succinylation modification in intrahepatic cholangiocarcinoma tissue is also an independent risk factor for the prognosis of patients with intrahepatic cholangiocarcinoma.High TKT-K319 succinylation modification activates platelets and is associated with greater platelet infiltration in intrahepatic cholangiocarcinoma tissue.