EfnB3 Promotes The Occurrence And Development Of Squamous Skin Cancer By Activating Notch1 And MAPK Signaling Pathways

Objective: Eph/Efn Bs signaling pathway acts as a bidirectional pathway with multiple links and multiple sites of action,playing an important role in regulating tumor microvessel formation,tumor microenvironment and immunity,tumor cell proliferation,migration,and invasion.Our previous experiments found that Efn B3 was abnormally elevated in clinical samples of skin squamous cell carcinoma.The purpose of this project is to further study the expression of Efn B3 in skin squamous cell carcinoma tissues and cell lines,and analyze its relationship with the clinic pathological parameters and survival time of patients with skin squamous cell carcinoma;to explore the effect of Efn B3 in vivo on DMBA/TPA-induced skin cancer,effects on proliferation,migration and invasion of skin squamous cell carcinoma in vitro;and to explore the mechanism of the effect in the process,to find new targets for the prediction of skin squamous cell carcinoma treatment and to find new biomarkers for the skin Squamous cell carcinoma provides theoretical basis and experimental basis for gene therapy.Methods: Part 1:(1)Efn B3 expression was analyzed in several squamous cells by q RT-PCR and Western Blotting experiments;(2)Head and neck squamous cell carcinoma(HNSC)data from the TCGA database was downloaded and analyze the relationship between Efn B3 expression level and different stages of skin squamous cell carcinoma from sample of patients in clinic;combined with clinical information in the database,the correlation between Efn B3 expression level and patient prognosis was analyzed;(3)The effect of Efn B3 on tumorigenesis and development was evaluated through the squamous cell carcinoma model induced by DMBA/TPA inwild type and Efn B3 knockout mice;(4)Efn B3-si RNA on A431 cells and transfect Efn B3 high expression plasmid was transfected on Hacat cells;(5)CCK-8 growth assay,flow cytometry,transwell migration and Matrigel invasion assays were performed to study the effect of Efn B3 on the proliferation,cell cycle,migration,and invasion of A431 cells.Part II:(1)The relationship between Efn B3 and Notch1 was analyzed by the information of TCGA clinical database;(2)The regulated relationship between Efn B3 and Notch1 pathways was verified by q RT-PCR and Western blot experiment.Part III:(1)Lable-free proteomics was used to comprehensively screen the altered proteins between knockout and wild-type mouse tumors.Reactome bioinformatics resources were used to reveal the biological functions and intracellular signaling pathways involved in Efn B3-regulated proteins are participated in.Some identified intracellular signaling pathways were validated by Western blotting experiments;(2)The anti-inflammation effect of Efn B3 was evaluated on TPA acute inflammation model in wild type and Efn B3 knockout mice.Results: Part 1: The protein and m RNA expression of Efn B3 in A431,Eca-109,and CNE cells are higher than that of Hacat cells;(2)The expression of Efn B3 was significantly related to TNM staging of patients with head and neck squamous cell carcinoma: as,the m RNA level of Efn B3 increased together with the increased grade of T stage(p <0.05).Besides,patients with highly Efn B3 expressed have a lower overall survival rate(p <0.05);(3)In the DMBA/TPA model,the absence of Efn B3 inhibits tumorigenesis and development of skin squamous cell carcinoma;(4)Compared with the negative interference group,the level of Efn B3 protein and m RNA levels were significantly reduced in si-Efn B3 cells(p <0.01);Compared with the control group,the Efn B3 protein and m RNA levels were significantly higher after transfect Efn B3 high expression plasmid(p <0.01);(5)After silencing Efn B3 in A431,cell proliferation was slowed(p <0.01),Cycle arrest was in G1 phase(p <0.05),cell migration and invasion ability were weakened(p <0.05).Part II:(1)TCGA database analysis showed that Efn B3 m RNA was positively correlated with Notch1 protein expression;(2)High expression of Efn B3 inhibited theexpression of NUMB protein,increase Notch1 intracellular protein NICD,transcription factor RPBJ-k,downstream target Hes-1 and H-Ras protein expression,activated Notch1 pathway;interference expressional of Efn B3 increases NUMB protein expression,inhibited Notch1 intracellular protein NICD,transcription factor RPBJ-k,downstream targets Hes-1 and H-Ras protein expression,inhibited Notch1pathway;in DMBA/TPA-induced mouse tumors,deletion of Efn B3 increased NUMB protein expression,inhibited Notch1 intracellular protein NICD,transcription factor RPBJ-k,downstream targets Hes-1 and H-Ras protein expression,and inhibited Notch1 signaling.Part III:(1)We identified 199 Efn B3 regulated proteins through quartic quantitative proteomic analyses of samples from knockout and wild-type mouse tumors.Bioinformatic analyses of Efn B3 regulated proteins showed that many proteins were involved in TCA cycle,platelet degranulation,MAP2 K and MAPK pathway activation,and the formation of translation initiation complexes,etc.Among them,Fga,Fgb,Fgg,Iqgap1,Actb,and Actg1 are involved in MAPK signaling pathway activation;(2)Western Blotting experiments have confirmed that deletion of Efn B3 leads to a significant decrease in protein expression of MAPKs family members:P-JNK(p <0.05),P-p38(p <0.05),and P-ERK(p <0.05),which is consistent with mass spectrometry results;Furthermore,tumor microenvironment-related inflammation proteins expression as COX-2,IL-1β,P-STAT3,p-65,P-p65,TNF-α in mouse tumors of lacking Efn B3 mice were significantly decreased(p <0.05);(3)In the TPA acute inflammation model,the deletion of Efn B3 inhibited the protein expression of P-STAT3,P-ERK,and COX2,and reduced the epidermal thickness caused by TPA.Conclusion: This study showed that Efn B3 is highly expressed in human skin squamous cell carcinoma tissues,and overexpression of Efn B3 is associated with poor prognosis in patients;Deletion of Efn B3 can inhibit tumorigenesis and development.Interfering with Efn B3 can slow cell proliferation,invasion and migration ability;Efn B3 may activate the Notch1 pathway and MAPK pathway to promote tumorigenesis and development through the TCGA database and Label-freeproteomics analysis.