| Background:Lung cancer is one of the most common malignant tumors and the leading cause of cancer deaths worldwide.Among them,lung adenocarcinoma accounts for about40% of all lung cancer cases.Although some achievements have been made in the research on the pathogenesis of lung adenocarcinoma(LUAD),and new methods have been developed in clinical treatment,lung adenocarcinoma is still one of the most aggressive and lethal tumor types.First,the overall survival period of the patient is less than 5 years.At least 50% of Asian lung adenocarcinoma patients have activating mutations in the epidermal growth factor receptor(EGFR)gene.Among them,the L858 R mutation of EGFR and the deletion of ΔE746-A750 account for about 85% of lung adenocarcinoma EGFR mutations.Although there are currently chemotherapeutics targeted for EGFR mutations,most patients develop new gene mutations to acquire drug resistance during the treatment process,resulting in unsatisfactory treatment effects.Therefore,further understanding of the pathogenesis of EGFR mutations and deepening the remodeling and research of EGFR mutation-related molecules will provide new targets for the clinical treatment of lung adenocarcinoma.Arl13 B is a member of the Ras GTPase family.It is located in the primary cilia and regulates the length of the cilia,as well as the transportation of vesicles,cell differentiation,cell movement and cytoskeleton.In recent years,studies have found that Arl13 B is involved in the occurrence and development of tumors,but the mechanism of Arl13 B in regulating tumors is still incomplete.Whether Arl13 B also functions through other signaling pathways remains to be studied.Objectives:We use mouse lung adenocarcinoma models and lung adenocarcinoma cell lines to confirm the regulatory effect of Arl13 B on the occurrence and development of lung adenocarcinoma;To further investigate the molecular mechanism by which Arl13 B regulates the EGFR signalling pathway to promote the occurrence and development of lung adenocarcinoma.To provide new targets for early diagnosis and treatment of EGFR mutation-induced lung adenocarcinoma.Methods:1.Determining that Arl13 B interacts with EGFR.The GST-Arl13B△19 and GST proteins were purified separately,and then combined with the Flag-EGFR fusion protein expressed by HEK293 T cells to perform GST pull-down experiments.2.Study the regulation of Arl13 B on the EGFR signaling pathway and determine the molecular mechanism of its regulation.(1)In order to further determine the molecular mechanism of Arl13 B affecting EGFR,we overexpressed Arl13 B in the 293 T gradient to detect the phosphorylation of downstream proteins in the EGFR signaling pathway.(2)Interfere with the expression of Arl13 B and detect the phosphorylation of downstream proteins in the EGFR signaling pathway.(3)A stable cell line overexpressing Arl13 B was constructed,and the regulatory effect of Arl13 b on EGF-induced EGFR endocytosis was tested.(4)A stable cell line with Arl13 B interference was constructed to detect the effect of Arl13 B interference on EGF-induced EGFR endocytosis.(5)Immunofluorescence experiment to detect the effect of overexpression of Arrl13 B on the localization of EGFR in endosomes.3.In vivo and in vitro experiments verify that Arl13 B knockout can inhibit the occurrence and development of lung adenocarcinoma induced by EGFR mutations.(1)Overexpressing or interfering with Arl13 B in lung adenocarcinoma cells,use Ed U test to detect changes in cell proliferation.(2)In order to further verify the regulation effect of Arl13 B on the occurrence and development of EGFR-induced lung adenocarcinoma in vivo,we constructed EGFR-L858 R and EGFR-L858R;Arl13B-ko gene mouse models,DOX induction,and observation of mouse lungs Partial tumor formation and survival time of mice.(3)Immunohistochemistry was used to detect the phosphorylation of EGFR and its downstream target proteins in mouse lung adenocarcinoma tissue,as well as the expression of MMP9 and KI67.Results:1.There is an interaction between Arl13 B and EGFR.(1)In 293 T cells,Flag-EGFR and GFP-Arl13 B are mutually immunoprec-ipitated.Endogenous EGFR protein can be immunoprecipitated with Arl13 B antibody,and endogenous Arl13 B protein can be immunoprecipitated with EGFR antibody,indicating that EGFR and Arl13 B can form protein complexes in cells.(2)Through the above research,it can be clear that Arl13 B is a new binding protein of EGFR,which provides a strong basis for elucidating the role and molecular mechanism of Arl13 B in the occurrence and development of tumors caused by EGFR mutations.2.Arl13 B promotes the activation of EGFR signaling pathway by regulating the endocytosis of EGFR.(1)Overexpression of Arl13 B in 293 T cells increases the phosphorylation level of target proteins downstream of the EGFR signaling pathway.(2)Interfering with Arl13 B,the phosphorylation level of EGFR downstream signal target protein decreases.(3)Arl13B can promote the activation of EGFR signaling pathway.(4)Arl13B regulates the activity of EGFR signaling pathway by regulating the endocytosis of EGFR.(5)It was found that in the absence of EGF,overexpression of Arrl13 B increased the localization of EGFR in the early endosomes;after stimulation with EGF,overexpression of Arrl13 B reduced the localization of EGFR in late endosomes.This result indicates that Arl13 B promotes the circulation of EGFR in the cell,and EGFR is repeatedly recovered to the cell membrane,thereby continuously activating the EGFR signaling pathway.(6)The above results indicate that Arl13 B realizes the repeated use of EGFR by promoting the circulation of EGFR in the cell,thereby continuously activating the EGFR signaling pathway.3.Arl13 B knockout can inhibit the occurrence and development of lung tumors caused by EGFR mutations.(1)Arl13B can not only affect the biological functions of EGFR wild-type lung adenocarcinoma cells,but also affect the biological functions of lung adenocarcinoma cells with EGFR activating mutations.(2)Arl13B knockout can slow down the occurrence and development of lung adenocarcinoma induced by EGFR mutation.(3)The immunohistochemical results of the lung tissues of genetic mice showed that after Arl13 B knockout,the activity of the EGFR signaling pathway decreased,and the proliferation and invasion ability of tumor cells decreased.Conclusion:In this study,we found that Arl13 B interacts with EGFR,regulates the endocytosis and cell membrane localization of EGFR,regulates EGFR signaling activity,and promotes the occurrence and development of lung adenocarcinoma. |
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