Molecular Mechanism Of The Interaction Between EIF4G1 And USP10 To Promote The Growth Of Non-small Cell Lung Cancer

ObjectiveLung cancer is one of the most common malignancies and poses a significant social and economic burden each year,with about half of new cases occurring in Asia.Lung Cancer can be divided into Non-Small Cell lung cancer and Small Cell Lung Cancer according to histological types,of which non-small cell lung cancer accounts for 80%-85%.Studies have found that the most common histological subtype of NSCLC(non-small cell lung cancer)is adenocarcinoma,followed by squamous cell carcinoma.Despite advances in surgery,radiotherapy,chemotherapy,molecular targeted therapy and immunotherapy for lung cancer,the average 5-year survival rate for lung cancer is only 19%,so the search for new specific diagnostic indicators and therapeutic targets remains important.Eukaryotic Translation Initiation Factor 4Gamma 1 EIF4G1(Eukaryotic Translation Initiation Factor 4 Gamma 1),an integral part of the initiation complex,recruits its binding proteins as scaffold proteins.Part of the EIF4 F complex,EIF4G1 is a protein scaffold that interacts with several components of the translation promoter and is required for translation.EIF4G1 is highly expressed in a variety of cancers and promotes tumor growth,such as nasopharyngeal carcinoma,inflammatory breast cancer,ovarian cancer,prostate cancer,lung cancer,malignant pleuromesothelioma,and so on.EIF4G1 also promotes tumor angiogenesis,malignant transformation,and phagocytosis.USP10(Ubiquitin-specific Protease 10)is a member of the USP family and is a deubiquitinating enzyme.USP10 has different effects in different tumors.USP10 is a carcinogen in breast cancer,glioblastoma,and prostate cancer,and a carcinogen suppressor in renal cell carcinoma,stomach cancer,and pancreatic cancer.The study also found that EIF4G1 interacts with USP10,but how they interact is unclear.Therefore,in this study,sh RNA and overexpressed plasmid were used to establish stable strains with USP10 knockdown and overexpression,and to study the regulatory effect of USP10 on EIF4G1 and its molecular mechanism,as well as whether USP10 affects the function of non-small cell lung cancer cells through the EIF4G1-dependent pathway.It provides a new target and therapeutic approach for the treatment of non-small cell lung cancer.MethodsThe methods of this study were as follows:(1)Stable strains with low knockdown and overexpression of USP10 were established by stable transfection technique.(2)Western blot technique was used to detect the influence of USP10 knockdown and overexpression on the expression level of EIF4G1 protein,observe the influence of USP10 knockdown and overexpression on the half-life of EIF4G1,and determine the influence of USP10 on the stability of EIF4G1.The effect of USP10 on EIF4G1 was detected after the cells were treated with MG132.(3)The effect of USP10 knockdown and overexpression on EIF4G1 m RNA expression was detected by RT-q PCR.(4)Immunocoprecipitation was used to detect whether USP10 exerted its deubiquitinating enzyme effect on EIF4G1.(5)In vitro cell experiments were conducted to explore whether USP10 affected the function of non-small cell lung cancer cells through EIF4G1 pathway,such as CCK-8 and clonal formation experiments to detect whether USP10 regulated the proliferation of non-small cell lung cancer cells through EIF4G1-dependent ways.Cell scratch assay was used to determine whether USP10 regulates non-small cell lung cancer cell migration in an EIF4G1-dependent manner.ResultsThe results of this study are as follows:(1)Western blot and RT-q PCR showed that the protein and m RNA expression levels of EIF4G1 were decreased in H1299 cells with USP10 knockdown;When USP10 is overexpressed,the protein and m RNA expressions of EIF4G1 are increased,and when USP10 is restored in USP10 knockdown cells,the expression of EIF4G1 is also recovered,which further indicates that USP10 has a positive regulatory effect on EIF4G1.(2)H1299 cells with knockdown and overexpression of USP10 were treated with the protein synthesis inhibitor actinobacillone(CHX),and the half-life of EIF4G1 was detected.The half-life of EIF4G1 was shortened when USP10 was knocked down,and the half-life of EIF4G1 was prolonged after USP10 was overexpressed,so USP10 increased the stability of EIF4G1.MG132 treatment of H1299 cells with USP10 knockdown showed that the expression of EIF4G1 could be restored,suggesting that USP10 affects the stability of EIF4G1 through the proteasome pathway.(3)In vivo ubiquitination experiments showed that USP10 deubiquitinated EIF4G1.(4)The proliferation and migration of H1299 cells with low USP10 knockdown were weakened,and the proliferation and migration of H1299 cells were restored after EIF4G1 was restored in H1299 cells with low USP10 knockdown.After overexpression of USP10,the proliferation and migration of NSCLC cells were enhanced.ConclusionsUSP10 plays a positive regulatory role on EIF4G1.USP10 can prolong the half-life of EIF4G1,making EIF4G1 more stable and not degraded.USP10 can increase the stability of EIF4G1 by reversing the ubiquitin-proteasome degradation pathway,and USP10 exerts its deubiquitinating enzyme effect on EIF4G1.USP10 enhances proliferation and migration of non-small cell lung cancer cells by stabilizing EIF4G1 expression.