| Oxaliplatin(OXA)resistance is a major obstacle to the treatment of liver cancer,and Epithelial-Mesenchymal Transition(EMT)has been shown to be closely related to OXA resistance.RNA binding motif 8A(RBM8A)is a core component of exon junctional complexes and is one of the proteins necessary for nonsense-mediated m RNA decay,is closely related with RNA transcription,translation,and cellular Cycle regulation and apoptosis regulation,and studies have confirmed that RBM8 A is widely involved in tumor development.Our previous study found that RBM8 A is highly expressed in hepatocellular carcinoma tissues and is associated with poor prognosis;high expression of RBM8 A could induce EMT in vitro,inhibit apoptosis of hepatocellular carcinoma cells,and promote whose proliferation,migration and invasion.However,it is unclear whether RBM8 A can participate in oxaliplatin resistance through induced EMT.If so,there is no research show its specific molecular mechanism.To further understand the role of RBM8 A in OXA resistance in hepatocellular carcinoma,and to elucidate the molecular mechanism of RBM8 A in the drug resistance network of hepatocellular carcinoma,and to identify potential drug targets for reversing the drug resistance,this study used RBM8 A knock-down and overexpressed-OXA-resistant model of hepatocellular carcinoma cells and the subcutaneous drug-resistant xenograft model in nude mice,through the detection of the changes of tumor-related functions,drug resistance and EMT characteristics,found the role of RBM8 A plays in OXA resistance in hepatocellular carcinoma through high-throughput microarray tech.And combined with bioinformatics to analyze and validate its potential downstream regulatory factors and signaling pathways.This research contains three parts.Chapter ? the preliminary role of RBM8 A in the resistance to oxaliplatin in hepatocellular carcinomaObjective:By constructing OXA-resistant model of hepatocellular carcinoma cells with low expression and overexpression of RBM8 A and subcutaneous drugresistant xenograft model in nude mice,the role of RBM8 A in cell function,drug resistance and growth of transplanted tumor were detected,and the correlation of RBM8 A and OXA resistance in hepatocellular carcinoma was preliminarily investigated.Methods:1.to establish drug-resistant hepatocellular carcinoma cell models BEL-7404/OXA and MHCC-97H/OXA(OXA concentration increasing method,intermittent induction),and by transfection to overexpression and RNA interference to establish a stable parental hepatocellular carcinoma cell model MHCC-97 H RBM8A-OE,BEL-7404-RBM8A-KD and OXA resistant hepatocellular carcinoma cell model MHCC-97H/OXA-RBM8A-OE,BEL-7404/OXA-RBM8A-KD;2.CCK-8 method was used to detect cell proliferation and IC50;3.Flow cytometry was used to detect the apoptosis and cell cycle.4.Western Blot was used to detect the changes of drug resistance-related proteins.5.Using BEL-7404/OXA-RBM8A-KD cells and BEL-7404/OXA-RBM8A-NC cells to construct a subcutaneous drug-resistant xenograft model in nude mice,and calculate the growth rate and mass size.Results:1.RBM8 A expression was significantly increased in drug-resistant hepatocellular carcinoma cells MHCC-97H/OXA,BEL-7404/OXA-compared with parental cells MHCC-97H-NC and BEL-7404-NC;2.proliferation and apoptosis of BEL-7404/OXA-RBM8A-KD cells increased,cells in G1 phase increased;proliferated and apoptosis of MHCC-97H/OXARBM8A-OE cells decreased significantly,cells in G1 phase reduced;3.The IC50 of BEL-7404/OXA-RBM8A-KD cells decreased,and the tolerance to OXA was significantly decreased.The IC50 of MHCC-97H/OXA-RBM8 AOE cells increased,and the tolerance to OXA was significantly increased;4.The expression levels of ABCG2,ABCB1,and ABCC1 were decreased in BEL-7404/OXA-RBM8A-KD cells.Increased expression levels of ABCG2,ABCB1,and ABCC1 were observed in MHCC-97H/OXA-RBM8A-OE cells;5.The growth rate and size of subcutaneous xenografts in nude mice constructed by BEL-7404/OXA-RBM8A-KD cells were significantly lower than those in BEL-7404/OXA-RBM8A-NC cells.Conclusions:The expression of RBM8 A is upregulated during the OXA resistance induction in hepatocellular carcinoma.Overexpression of RBM8 A promoted the proliferation of parental and drug-resistant hepatocellular carcinoma cells in vitro,reduced the proportion of apoptosis and G1 phase cells,induced the expression of drug-resistant proteins,and enhanced tolerance to OXA suggests that RBM8 A is involved in the process of OXA resistance in hepatocellular carcinoma cells.Chapter ? investigation on RBM8 A Mediated EMT in regulation the Mechanism in the Resistance to OXA in Hepatocellular carcinomaObjective:RBM8A silenced and overexpressed-OXA-resistant hepatocellular carcinoma cell model and nude mouse subcutaneous xenograft model were established,the changes of cytoskeleton,EMT characteristics,cell migration and invasion ability after RBM8 A alteration were detected to investigate RBM8A-mediated EMT mechanism in resistant to OXA in hepatocellular carcinoma.Methods:1.cytoskeletal staining were to detect changes in morphology change in RBM8 A overexpressed and silenced hepatocellular carcinoma cells(MHCC-97H-RBM8A-OE,BEL-7404-RBM8A-KD)and OXA-resistant with RBM8 A overexpressed and silenced cells(MHCC-97H/OXA-RBM8A-OE,BEL-7404/OXA-RBM8A-KD)2.Western Blot and immunofluorescence were used to detect the expression of EMT factors;3.Scratch method and Transwell to detect changes in cell migration and invasion ability;4.In the subcutaneous drug-resistant xenograft model of nude mice,immunohistochemistry and Western Blot were used to detect the expression of EMT and drug resistance proteins in tumor tissues.Results:1.Cytoskeletal staining results showed that the BEL-7404/OXA-RBM8A-KD cells' cytoskeleton were more prone to round,less pseudopod epithelial morphology;whose of MHCC-97H/OXA-RBM8A-OE cells were more prone to fusiform interstitial form.2.Western Blot and cytofluorescence assay showed that the expression of Ecadherin was significantly increased in BEL-7404/OXA-RBM8A-KD cells,and the expression levels of N-cadherin and snail were significantly decreased.The expression level of E-cadherin was significantly decreased in MHCC-97H/OXA-RBM8A-OE cells,and the expression levels of N-cadherin and snail were significantly increased.3.BEL-7404/OXA-RBM8A-KD cells have decreased migration and invasion ability;those of MHCC-97H/OXA-RBM8A-OE cells enhanced.4.The expression of E-cadherin in subcutaneous xenografts of nude mice constructed with BEL-7404/OXA-RBM8A-KD cells was significantly higher than those constructed by BEL-7404/OXA-RBM8A-NC cells,and the expression of N-cadherin and snail was significantly lower,the expression levels of multidrug resistance proteins ABCG2,ABCB1,and ABCC1 were significantly lower in BEL-7404/OXA-RBM8A-NC cells than in BEL-7404/OXA-RBM8A-NC cells.Conclusions:Overexpression of RBM8 A induces the interstitial phenotype of parental and OXA-resistant hepatocellular carcinoma cytoskeleton,inhibits the expression of epithelial cell marker E-cadherin,promotes the expression of stromal cell markers N-cadherin and Snail,and enhances the cell migration and invasion.RBM8 A can participate in hepatocellular carcinoma resistance to OXA by inducing EMT.Chapter ? investigation on screening molecular in hepatocellular carcinoma resistance to OXA mechanism based on high-throughput microarray and bioinformaticsObjective:Through high-throughput microarray and bioinformatics,we analyzed the differentially expressed gene network in RBM8 A silenced and overexpressed OXA-resistant hepatocellular carcinoma cell,and combined with in vitro verification on the selected regulatory factors and signaling pathways to explore the RBM8A-related molecular network of hepatocellular carcinoma resistance to OXA.Methods:1.High-throughput microarray technique was used to screen differentially expressed genes in cells MHCC-97H/OXA-RBM8A-OE and BEL-7404/OXARBM8A-KD;2.differentially expressed genes were analyzed by weighted correlation network analysis(WGCNA)and clustering into gene modules with coexpression phenomenon;3.To identify potential regulatory factors and signaling pathways by enrichment analysis and hypergeometric analysis.q RT-PCR and Western Blot were used to verify the expression levels of differential genes and regulatory factors in vitro.Results:1.Venn diagram shows the result of cell differential gene intersection between BEL-7404/OXA-RBM8A-NC cells and BEL-7404/OXA-RBM8A-KD cells,as well as MHCC-97H/OXA-RBM8A-NC cells and MHCC-97H/OXA-RBM8 AOE cells,8365 identical differential genes were obtained,q RT-PCR and Western Blot assays confirmed that IGF1,MAPK4 and S100A1 were significant differentially expressed genes.2.WGCNA analysis showed that the differential genes showed significant coexpression and clustered into five modular systems.3.GO function and KEGG pathway enrichment analysis showed that 18672 biological processes,2639 cells,3932 molecular functions and 943 KEGG pathways involved.The KEGG pathway mainly includes cell adhesion molecules,cell cycle,hepatitis B virus carcinogenesis,actin cytoskeleton regulation,RNA degradation and transport,m RNA monitoring pathway,RIG-Ilike receptor signaling pathway,Erb B signal Pathway,insulin signaling pathway,MAPK signaling pathway and PI3K-Akt signaling pathway.4.Hypergeometric analysis combined with q RT-PCR and Western Blot validation showed that Lnc RNA MALAT1,Lnc RNA FENDRR,STAT3 and HDAC9 are potential core regulators.Conclusions:RBM8A has extensive transcriptional regulation in the process of resistance to OXA in hepatocellular carcinoma cells.RBM8 A may induce EMT through key regulatory factors such as Lnc RNA MALAT1,Lnc RNA FENDRR,STAT3 and HDAC9,as well as insulin signaling pathway,MAPK signaling pathway and PI3K-Akt signaling pathway.And participate in the process of hepatocellular carcinoma resistant to OXA. |
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