The Role And Mechanism Of RPS6 In Cholangiocarcinoma

Background & Aim: Cholangiocarcinoma is a malignant tumor originating from bile duct epithelial cells,and is characterized by difficult early diagnosis and poor prognosis.In the early part of this paper,based on iTRAQ proteomics technology and HCS proliferation screening experiment,it was found that RPS6 is highly expressed in cholangiocarcinoma tissues and silencing RPS6 can significantly inhibit the proliferation of cholangiocarcinoma cells.Some studies have found that RPS6 is abnormally highly expressed in various tumors.The potential regulatory role is reflected in the development,further study of its biological function and relationship with cholangiocarcinoma,further study on its biological function and its relationship with cholangiocarcinoma,explore its possible molecular signaling pathways regulating cholangiocarcinoma growth,it will provide a theoretical basis for the prognosis evaluation and molecular targeted therapy of patients with cholangiocarcinoma.The aim of this study was to explore the molecular biological mechanism of RPS6 regulating the proliferation of cholangiocarcinoma cells by analyzing the relationship between the expression of RPS6 in the tissues of cholangiocarcinoma patients and the prognosis of patients.Methods: iTRAQ proteomics was used to identify the protein profile changes of patients with cholangiocarcinoma,20 differential proteins were selected and the genes that significantly inhibited the proliferation of cholangiocarcinoma cells were selected by high content proliferation experiment RPS6,immunohistochemical detection of RPS6 and its phosphorylated form p-RPS6 expression in cholangiocarcinoma and survival analysis.Construct a lentiviral interference plasmid,verify the knockdown effect by qRT-PCR and Western Blotting,CCK-8 cell proliferation experiment to verify the inhibitory effect of RPS6 knockdown on the growth of cholangiocarcinoma cells.Finally,transcriptomics high-throughput sequencing technology was used to detect the changes in the expression level of the whole transcriptome of cells after down-regulation of RPS6 gene,and qRT-PCR was used to verify the changes in differential gene expression levels.Results: A total of 786 differential proteins were detected based on iTRAQ-based proteomics technology,and the gene of the experimental group with positive proliferation screening test was RPS6,and its proliferation fold changes reached 3.41 times.The results of immunohistochemical staining and survival analysis of tissue sections showed no significant difference in overall survival and relapse-free survival between patients with high and low expression of RPS6(OS,P = 0.131;RFS,P = 0.108),while p-RPS6 was highly expressed Compared with patients with low expression,overall survival was worse,and relapse-free survival was shorter(OS,P = 0.018;RFS,P = 0.007).CCK-8 cell proliferation experiments confirmed that downregulating RPS6 expression can significantly inhibit the proliferation of cholangiocarcinoma cells RBE and QBC939.The research strategy of high-throughput sequencing analysis based on transcriptomics illustrates that the main signal pathways of RPS6 regulating cholangiocarcinoma are DNA replication,Cell Cycle,p53 Signaling pathway,and further confirmed by qRT-PCR that after knockdown RPS6,MDM2,TP53 and CDKN1 A genes were significantly up-regulated,and cyclindependent kinases CDK2,CDK4 and CDK6 genes were down-regulated.Conclusion: RPS6 and p-RPS6 are highly expressed in cholangiocarcinoma tissue,and the high expression of p-RPS6 is significantly related to the OS and RFS of cholangiocarcinoma patients.Down-regulation of RPS6 can significantly inhibit the proliferation of cholangiocarcinoma cells,the mechanism may be through the MDM2-p53 signaling pathway inhibits the cell cycle to affect the proliferation of cholangiocarcinoma cells.RPS6 may be a potential target for the treatment of cholangiocarcinoma.