Role And Mechanism Of RMP In Regulating Anti-Oxidation Ability And Drug Resistance Of Cholangiocarcinoma

Background and purposesCholangiocarcinoma is a malignant tumor that originates from the epithelium of the liver biliary system.In all primary liver tumors,the incidence rate of Cholangiocarcinoma is 10%-15%,which is increasing year by year.Cholangiocarcinoma is a higher degree malignancy characterized by late diagnosis and poor prognosis.Statistics show that the five-year survival rate is only 30%-40%,and the median survival time of unresectable patients is only 12-15 months.East Asia and South Asia are areas with high incidence of cholangiocarcinoma in the global perspective.Therefore,it is particularly important to study the mechanism of its occurrence and development.Since 1998,Japanese researchers discovered that RMP?RNA polymerase II subunit5-mediating protein,also called URI,Unconventional prefoldin RPB5 Interactor?,people's understanding of RMP has incresed.mTOR phosphorylates RMP,inhibits the expression of related genes,and assists yeasts and cells to survive the harsh nutrient environment.In ovarian cancer research,it was found that RMP binds to PP1?to form a complex and inhibits its phosphatase activity,thereby destroying the negative feedback pathway consisted of PP1?and S6K1-BAD which strengthened BAD phosphorylation,thus apoptosis is reduced,and survival is promoted.In addition,RMP has also been reported to promote tumor growth in various tumors such as prostate cancer,multiple myeloma and endometrial cancer.Our group also explored the role of RMP in liver cancer,and found that RMP can facilitate the binding of p65 to the IL-6 promoter region,promote its transcription,and produce more IL-6 in liver cancer cells,thus activating IL-6/STAT3signaling pathway.The STAT3 signaling pathway promotes liver cancer metastasis and self-renewal.However,the research on the mechanism of RMP in cholangiocarcinoma has been rarely reported,and further research and exploration are urgently needed.Changes in internal and external environment play an important role in tumor growth.On one hand,cell genetic mutation caused by ROS?reactive oxygen species?is an important cause of tumorigenesis.On the other hand,the large amount of ROS production and release causes the breakdown of intracellular redox equilibrium,leading to a series of DNA damage,cell membrane structure destruction,cell organelle damage and other phenomena eventually lead to the occurrence of apoptosis.In recent years,studies have shown that ROS production is inhibited during tumorigenesis.Therefore,it is of great significance to explore the mechanism of ROS during tumor development.The KEAP1-NRF2-ARE signaling pathway plays an important role in the antioxidant process of cells.KEAP1?Kelch-like ECH-associated protein 1?provides a scaffold for the E3 ubiquitin ligase CUL3,which mediates NRF2 degradation by ubiquitin-proteasome,allowing NRF2 to remain only low to ensure daily life activities.In oxidative stress,upstream related stimuli mediate NRF2 degradation and terminate into nuclear,promote the transcription of antioxidant-related genes,and participate in various physiological activities related to cellular antioxidants.This study intends to comprehensively and deeply study the regulation of RMP on cholangiocarcinoma cells biological behaviors and oxidative stress process by establishing a variety of differentially expressed RMP cell lines using RMP overexpressing or interfering lentiviruses,mouse spontaneous cholangiocarcinoma model,mouse tumor-bearing model and tail vein injection lung metastasis model combining with clinical cholangiocarcinoma samples.We also purposed on elucidating the role of RMP in signal transduction further,verifying its value as a target for interventional therapy of cholangiocarcinoma and improving tumor chemotherapy resistance.Methods1.Establish RMP stably overexpressed or deleted Cholangiocarcinoma cell lines by lentivirus.2.Compare the cell biology behaviors of Cholangiocarcinoma cells by CCK8proliferation assays,Transwell migration assays,plate colony formation assays,soft argar colony formation assays.3.Establish subcutaneous tumor model and lung metastasis model to observe the differences in tumor formation,growth,migration abilities of RMP differentially expressed ICC cells.4.Real-time PCR and protein electrophoresis detect changes in mRNA and protein levels of antioxidant-related genes from transcription and translation levels,respectively.5.Detect the differential expression of GSSG and GSH in RMP cells and tumor tissues.6.Observe the effect of RMP high and low expression on the transcriptional activity of antioxidant response elements by luciferase reporter system.7.Flow cytometry was used to detect the effect of RMP on ROS and apoptosis.8.Construction of truncated plasmids with different functional domains of RMP and KEAP1 and RMP mutant plasmids.9.Co-immunoprecipitation was applied to observe the interaction of KEAP1 protein with NRF2 and RMP proteins to identify their relationships and interaction sites.10.Immunofluorescence was used to observe the localization of KEAP1,RMP and NRF2 proteins in cells.11.Crystal violet staining was used to detect the concentration-growth curve of RMP differentially expressed cells under chemotherapeutic drug oxaliplatin.12.To observe the effect of oxaliplatin on the tumor growth of the subcutaneous tumor-bearing model of nude mice with differential expression of RMP cells.13.Establish hepatocyte-specific RMP knockout mice and cross-breed mice with PTEN^flox/+;Kras^G12D;Alb-Cre spontaneous cholangiocarcinoma model to compare the effects of RMP knockout on the occurrence and progression of intrahepatic cholangiocarcinoma in mice.14.Isolate and establish of primary cells of cholangiocarcinoma mouse model and compare difference in antioxidant-related signaling pathways.15.Detect the expression of RMP,anti-oxidation,proliferation,injury and other marker proteins in tumor tissues by immunohistochemistry.16.Analyze RMP expression in clinical intrahepatic cholangiocarcinoma samples and its relationships with clinical relevance,overall survival,and tumor-free survival.17.Intrahepatic cholangiocarcinoma PDX samples were used to observe the effect of RMP expression on tumor growth.Results1.Southern blotting confirmed that flox was correctly inserted into the Exon3 of mouse RMP gene by 3'and 5'two-way probes and liver-specific RMP knockdown was achieved in RMP^flox;Alb-Cre mice.2.Knocking out of RMP on the basis of AKPP spontaneous cholangiocarcinoma mouse model slowed down the occurrence and development of cholangiocarcinoma.3.RMP promoted anchorage-independent growth of cholangiocarcinoma cells.4,RMP can enhance oxidative stress tolerance in cholangiocarcinoma cells and increase apoptosis resistance.5,RMP high and low expression in cholangiocarcinoma cells and tumor tissues affected the anti-oxidative capacity by regulating the expression of NRF2 protein levels,while RMP had no effects on the expression of NRF2 transcriptionally.6.The degradation rate of NRF2 protein was accelerated when RMP was interfered;while stabilities of NRF2 protein was increased in RMP overexpressed cells.7.RMP competitively bounded to the Kelch region in the KEAP1 protein through the D2 region,which reduced the degradation of NRF2 protein and increases its stability.8.Increased expression of RMP increased the resistance of cholangiocarcinoma cells to oxaliplatin.9.The overall survival and disease-free survival of patients with intrahepatic cholangiocarcinoma with high RMP expression were shorter than those with low RMP expression and the prognosis was relatively poor.10.RMP was positively correlated with NRF2 expression in intrahepatic cholangiocarcinoma and patients had negative prognosis with both RMP and NRF2overexpressed.11.The high and low expression of RMP in PDX samples of intrahepatic cholangiocarcinoma affected tumor growth rate.12,Differential expression of RMP in intrahepatic cholangiocarcinoma PDX primary cells had different antioxidant capacities.ConclusionThis study found that the oncogene RMP enhances the anti-oxidation ability,resistance to apoptosis and promotes anchorage-independent growth and enhances tumor cell resistance to chemotherapy drug oxaliplatin of cholangiocarcinoma cells for the first time.In mouse spontaneous cholangiocarcinoma model,RMP knocking down inhibits tumorigenesis and progression;In addition,RMP promotes subcutaneous growth and lung metastasis of cholangiocarcinoma in nude mice.RMP is associated with NRF2 expression in clinical intrahepatic cholangiocarcinoma cohorts and patients with high RMP and or NRF2 expression have a poor prognosis.The above achieved by the following molecular mechanism:RMP competitively binds to the Kelch region in the KEAP1 protein molecule through the D2 region of its protein molecule,which reduces the degradation of NRF2protein and increases its stability,thereby promoting the expression of downstream related antioxidant molecules and increasing cellular antioxidant abilities.This study further broadens the role of RMP in tumorigenesis and development,and firstly describes the important role of RMP in the anti-oxidation process of cholangiocarcinoma and provides a direction for improving the therapeutic effect of chemotherapy drugs and improving drug resistance.