The Function And Mechanism Of RNF43 Site Mutations In The Oncogenesis And Development Of Gallbladder Carcinoma

Background and Purposes Gallbladder cancer ranks sixth in the incidence of digestive tract malignancy.Its mortality rate is close to the incidence rate since its extremely poor prognosis.Gallbladder cancer is lack of specific clinical manifestations in early stage.Nowadays,radical surgery is the only curable therapy,but there is a rate of postoperative recurrence and metastasis.Gene mutations play an important role in tumorigenesis.The study of tumor-driven genes not only reveals the causes of tumors,but more importantly it can be used as an entry point to explore the treatment.As an inhibitor of Wnt signaling pathway,Ring finger protein 43(RNF43)plays an important regulatory role in individual development,cell proliferation and differentiation.It has a high frequency of mutation in gallbladder carcinoma and has a close relationship with the occurrence of gallbladder carcinoma.It is worth further study.Methods1.Through the TOPflash/FOPflash dual luciferase reporter gene system assay and qPCR to detect the transcriptional changes of target genes downstream of the Wnt pathway,to confirm the inhibitory effect of RNF43 on the Wnt pathway,and the effect of the Y332* point mutation on RNF43 function.2.To explored the effect of protein RNF43 and its mutant Y332* on the proliferation and invasion ability of gallbladder cancer cells in vitro.3.Protein cross linking,tandem affinity purification and LC MS/MS were used to determine the interacting protein of RNF43,and to verify the effect of RNF43 point mutation Y332* on protein interaction by CO-IP.4.The expression of RNF43 in 40 cases of gallbladder carcinoma and its adjacent tissues was detected by Real-Time qPCR.The prognosis and clinicopathological information of the patients were analyzed comprehensively to explore the relationship between functional changes of RNF43 and the development and the prognosis of gallbladder carcinoma.Results1.RNF43 inhibits Wnt signaling pathway in gallbladder cancer cells;2.The inhibitory effect of the RNF43 truncation mutation Y332* on the Wnt signaling pathway is partially lost,which may be related to its DVL binding site deletion;3.In vitro,we found that RNF43 and point mutation Y332* had no significant effect on the proliferation and migration of gallbladder cancer cell lines;4.It was found that RNF43 can interact with the protein PRMT5,whereas the Y332*mutation impairs its binding ability;5.The differential expression of mRNA in gallbladder carcinoma showed that the expression level of RNF43 was not significantly different between cancer and adjacent tissues;6.The expression level of RNF43 was not significantly correlated with the development of gallbladder carcinoma and the prognosis of patients.Conclusion Although studies have shown that RNF43 is not sufficiently related to the development of gallbladder cancer,the role of RNF43 loss of function in the malignant process of gallbladder epithelium cannot be ruled out.