The Prognostic Value And Biological Role Of EIF3b In Clear Cell Renal Cell Carcinoma

IntroductionRenal cell carcinoma is the most common malignant tumor in urologic cancers,with a increasing incidence in recent years.Renal clear cell carcinoma(ccRCC)is themain type of renal cell carcinoma,accounting for about 75%.It is also the cancer with worst prognosis and the highest fatality rate.Once ccRCC metastasizes,its five years survival rate will be less than 9%.Although clinical surgical develops in recent years has improved the prognosis,the longtime survival rate remains unsatisfactory.Therefore,to clarify the underlying molecular mechanism mediating migration and invasion of renal clear cell carcinoma is the urgent problem of the current research and treatment.Many recent studies have shown that abnormal protein synthesis plays an important role in tumor development and progression.More importantly,transcription initiation process is the key speed limit process of the protein synthesis.Family of transcription initiation factors(eIFs)is a group of key factor mediating mRNA translation.Among those twelve known eukaryotic transcription factors,eIF3 is in the largest and most important subunits complex and eIF3b is the core of eIF3.It is the major scaffold protein and the other subunits have different affinity of combining with eIF3b.This eIF3 platform can recruit other proteins to form pre-initiation complex 43s.In addition,eIF3b can interact with eIF4G,forming pre-initiation complex 48s which can combine with start codon AUG complex.Studies have shown that that eIF3 subunits,including eIF3a,eFI3b,eIF3h,eIF3i and eIF3m,are a variety of potential prognostic molecular cancer markers which are more likely to be detected.And in the future,they are likely to be the cornerstone of a new class of cancer treatment therapeutic methods.Among them,eIF3b is reported to be a prognostic factor in malignant glioma,colon cancer,esophageal squamous cell carcinoma and bladder cancer.However,the prognostic value and the biological role of eIF3b in ccRCC are still not clear.In order to understand the of eIF3b biological role and its underlying molecular mechanisms,this research is going to be carried out using proteomics and transcriptomics,with combination of many cell biological technologies,include clinical research,cell experiments and animal experiments.Ultimately,we will uncover the role of eIF3b in ccRCC and its prognostic significance,which could provide theretical basis for the research on cnacer initiation and progression as well as targeted therapy.Part One:The Expression Level and Prognostic Value of eIF3b in ccRCC-eIF3b is Independently Prognostic for ccRCC PatientsObjectives1.Evaluate eIF3b expression level in ccRCC tissues,its relationship with clinicopathological parameters;2.To investigate its clinical prognostic value.Methods1.Collect clinical specimens,detecting eIF3b expression level in ccRCC using western blotting and immunohistochemistry(IHC)technologies;2.Using correlation analysis to detect eIF3b correlation with other clinical parameters;3.Univariate and multivariate analyses of eIF3b with prognosis.Results1.In 82 cases of ccRCC patients,45 cases of patients are tuor-eIF3b positive,and eIF3b are expressed in the cell nucleus and cytoplasm.EIF3b protein expression level was positively related with Furhman nuclear grade(p = 0.044),without obvious correlation with TNM staging;2.Kaplan Meier survival analysis showed that high eIF3b protein expression,higher TNM staging and higher Furhman nuclear grading was associated with a worse prognosis.COX multi-factor analysis showed that tumor eIF3b protein expression levels and TNM staging is independent prognostic factors in patients with ccRCC.Conclusion1.eIF3b plays a role in ccRCC progression and it can serve as a prognostic marker for ccRCC patients.More eIF3b expression,worse prognosis for patients will develop.Part Two:The Biological Role of eIF3b in ccRCC-eIF3b Depletion Inhibit Cell Proliferation,Migration,InvasionObjectives1.To explore and discuss biological role of eIF3b in ccRCC;2.Explore the feasibility of eIF3b as a targeted therapeutical target,uncovering the role of eIF3b for ccRCC treatment.Methods1.Applying of small interference RNA technology to knock down eIF3b expression in kidney cancer cell lines,which are 786-o and A498 cells;2.The cell proliferation assay and colony formation experiment detect eIF3b prohibited proliferation;3.Cell scratch experiment and Transwell assay knock detected a reduced invasion and migration ability of eIF3b-deleted cells;4.Subcutaneous formation experiment of Nude mice is used to explore change of tumor formation ability.Results1.After eIF3b knocked down with small interference technique,cell proliferation,cell migration and invasion were inhibited;2.eIF3b knockdown inhibited the growth of subcutaneous xenografts in nude mice.Conclusions1.In vitro experiment showed depletion of eIF3b can inhibit the proliferation,migration and invasion ability;2.Animal experiments suggest eIF3b is a potential therapeutic target for ccRCC patients.Part Three:The Mechanism Researchon How Tumor eIF3b Functions——eIF3b contributes to tumor malignancies through regulation of EMTand Akt signaling pathwayObjectivesExplore and discuss potential underlying mechanisms of elF3b regulating cell proliferation,cell cycle,and cell apoptosis in ccRCC.Methods1.Flow cytometry technology to detect change of cell cycle and apoptosis of eIF3b-deleted cells;2.Western blotting were used to test cycle apoptosis related proteins.to verify the flow changes of cell cycle and apoptosis;3.Using protein electrophoresis experiment to test changes of proteins involved in epithelial to mesenchymal transformation(EMT),to verify cell migration andinvasion change results;4.Check the change of Akt pathways after eIF3b deletion,which are the upstream pathways of cell proliferation,cell cycle and cell apoptosis;5.Clarify EMT upstream pathway,?-catenin pathway,to explore the molecular mechanism of cell invasion and migration changes.Results1.After knocking down eIF3b protein expression,progression of the cell cycle is inhibited,including G1/S arrest and G2/M arrest.After knocking down eIF3b expression,changes of proteins involved in the G1/S and G2/M progression verified this cell cycle arrest;2.After eIF3b knocked down,more apoptotic cells are detected using flow cytometry technology.Results of western blotting of apoptotic proteins verified this increased apoptosis in protein level;3.After eIF3b knocked down,epithelial marker,E-cadherin,is up-regulated and EMT inhibitors,Slug and Snail,are down-regulated;Mesenchymal markers,N-cadherin and Vimentin,are down-regulated.In addition,EMT upstream regulatory protein,?-catenin,is significantly reduced and its targets,Cyclin D,is also reduced,indicating that downregulation of P-catenin pathway is the reason of EMT suppression;4.Western blotting results of proteins involved in Akt pathways show that,after eIF3b deletion,Akt pathways are suppressed,including integrin/FAK/Akt pathway which regulate cell cytoskeleton and cellular adhesion,Akt/mTOR/HIF pathway which regulate cell proliferation,apoptosis and cell survival,and Akt/GSK-3?pathway which regulate cell apoptosis.Conclusions1.Inhibition of eIF3b expression can inhibit cancer cell proliferation by causing cell cycle arrest and promoting cell apoptosis;2.Reduced expression of eIF3b can suppress EMT progression through inhibiting ?-catenin pathway,which suppressed cell invasion and migration;3.After knock down of eIF3b,the inhibition of Akt pathway,include integrin/FAK/Akt pathway,Akt/mTOR/HIF pathway and Akt/GSK-3? pathway,was the main reason of cell biology changes.Part Four:Prognostic value of eIF3b expression in adjacent normal kidney tissues(ANK)——eIF3b in ANK work as a tumor-suppressive protein Objectives1.Evaluate eIF3b expression level in adjacent normal kidney tissues(ANK),its relationship with clinicopathological parameters;2.To uncover its clinical prognostic value;3.To explore the relationship between the tumor eIF3b and ANK eIF3b.Methods1.Collect clinical specimens,detecting eIF3b expression level in ANK using using real-time fluorescent quantitative PCR(RT-PCR),protein electrophoresis,western blotting and immunohistochemistry(IHC)technologies;2.Using correlation analysis to detect ANK eIF3b correlation with other clinical parameters;3.Univariate and multivariate analyses of ANK eIF3b with prognosis.Results1.Compared with tumor tissues,the expression of eIF3b in ANK is up-regulated(p=0.04)and it is correlated with T grade of tumor;2.Kaplan-Meier overall survival analysis showed high-expression of ANK eIF3b is correlated with good prognosis(p=0.047).ConclusionANK eIF3b may be play a role in protect the normal tissue from the tumor,which is totally different from tumor eIF3b.