Molecular Mechanism Of RNF41 Induces TTC1 Ubiquitination And Activates Ras-ERK Signaling Pathway To Promote Proliferation And Metastasis Of Cholangiocarcinoma

Objective: The aim of this study was to investigate the role of Ring finger protein 41(RNF41)in the development of cholangiocarcinoma and to elucidate the molecular pathways and underlying mechanisms required for this effect to provide strategies for enriching the treatment of cholangiocarcinoma.Methods: The expression of m RNA and protein of RNF41 in tumor tissues and adjacent normal tissues of patients with cholangiocarcinoma was analyzed by bioinformatics,Western blotting and immunohistochemistry;Clinical data and bioinformatics analysis of the effect of RNF41 on the prognosis and survival of patients with cholangiocarcinoma and the relationship between tumor clinical characteristics;After transfection of RNF41 si RNA into HCC9810,RBE and HUCCT1 cells,the proliferation of cholangiocarcinoma cells by RNF41 was analyzed using CCK-8,Ed U,colony formation assay and Western blottin assay;The migration and invasion of cholangiocarcinoma cells by RNF41 were further analyzed using transwell assay,scratch assay and marker protein assay,and the effect of RNF41 on epithelial-mesenchymal transition of cholangiocarcinoma cells was detected by Western blotting assay;The effect of RNF41 on tumor growth in nude mice was analyzed by tumor formation assay,Western blotting assay and immunohistochemical staining;Subsequently,mass spectrometry and immunoprecipitation techniques were used to detect the factors interacting with RNF41.In vitro ubiquitination assay,Western blotting assay,immunoprecipitation,and immunofluorescence co-localization were used to deeply explore the potential molecular regulatory mechanism of RNF41 in cholangiocarcinoma.Results: Bioinformatic analysis of RNF41 m RNA and protein levels showed that the expression level of RNF41 in cholangiocarcinoma tumor tissues was significantly higher than that in corresponding adjacent non-cancerous tissues.Bioinformatics analysis of clinical cases showed that the increased expression of RNF41 was closely related to the histological grade of CHOL and the stage of tumor lymph node metastasis,and the high expression of RNF41 was closely related to the poor prognosis of CHOL patients.Through in vitro cell assay,we found that RNF41 could promote tumor cell proliferation,migration,invasion and cell EMT.However,low expression of RNF41 could inhibit the subcutaneous tumorigenicity of cholangiocarcinoma cells in nude mice,and the results of EMT-related protein were consistent with those of in vitro cell assay.Mass spectrometry combined with immunoprecipitation analysis revealed that RNF41 interacted with Tetratricopeptide repeat domain 1(TTC1).Immunoprecipitation and Western blotting experiments showed that RNF41 stabilized TTC1 protein by promoting its K6-linked polyubiquitination.Mechanistic studies by Western blotting showed that phosphorylated RAF,ERK,or MEK levels were significantly upregulated in RNF41 OE HCC9810 cell,which were reversed by TTC1 si RNA treatment,and TTC1 silencing partially reversed the effects of RNF41 overexpression on proliferation and migration.Conclusion(s): RNF41 can promote the occurrence and development of cholangiocarcinoma and is closely related to clinicopathologic features such as tumor stage,lymph node metastasis and poor prognosis of patients;Low expression of RNF41 can inhibit the proliferation,migration,invasion,EMT and tumor formation ability of cholangiocarcinoma cells;RNF41 is involved in the occurrence and development of CHOL by mediating the ubiquitination regulation of Ras/ERK signaling pathway of TTC1.