Effect Of MAPK3 On Invasion And Metastasis Of Esophageal Squamous Cell Carcinoma

Background:Esophageal cancer is a malignant tumor occurring in esophageal epithelium,in China,esophageal squamous cell carcinomas(ESCC)and esophageal adenocarcinoma(EAC)constitutes the most pathological type of esophageal cancer,which ESCC patients account for about 90%.Due to the characteristics of invasive growth and metastasis,the 5-year survival rate of esophageal cancer is less than 20%.In recent years,proteomics has been widely studied in cancer,and its related applications have shown an important role in cancer research.Since there are few studies related to ESCC,the study conducted proteomic experiments on ESCC and para-carcinoma tissue samples to screen variationally expressed proteins,and combined with bioinformatics analysis,found the key genes:mitogen-activated protein kinase 3(MAPK3).Studies have shown that MAPK3 gene is related to breast cancer and lung cancer.The MAPK3 gene is variationally expressed in various tumors,such as prostate cancer and liver cancer,and is involved in the formation and progression of tumor.At present,there are few studies on MAPK3 gene in ESCC,and it may play an important functional role in ESCC.Methods:1.A total of 27 pairs of ESCC tissues and para-carcinoma tissue samples were collected from the Department of Thoracic Surgery of Sichuan Provincial People’s Hospital from May to November 2019.None of the patients received preoperative radiotherapy,chemotherapy or other anticancer treatments;2.The collected samples were screened for variationally expressed proteins by mass spectrometry,and the potential key genes were identified by further bioinformatics analysis;3.The expression levels of key genes in ESCC and para-carcinoma tissues were tested by immunohistochemistry(IHC).4.CRISPR/Cas9 gene editing technology was used to construct key gene knockout ESCC,and stable key gene knockout cell lines were obtained through screening and identification of monoclonal tumor cells;5.ESCC of organoids with key gene knockout and carry out proliferation trans-well experiment;6.ESCC transplantation model was constructed to observe tumor formation in the mice,and hematoxylin-eosin(HE)staining was performed on tumor tissues to observe the level of cell differentiation.Results:1.The results of mass spectrometry showed that 4967 proteins were identified in proteomics experiment,and a total of 1200 variationally expressed proteins were identified,among which 320 were up-regulated and 880 were down-regulated.2.Bioinformatics analysis of differentially expressed proteins screened out the key genes:CDC5L,VCL,ACTB,MAPK3,ALB,ATP5 B,CYCS,CAT,ATP5A1.Protein-protein interaction network map,the key gene MAPK3 was found;3.The expression level of MAPK3 gene in ESCC tissue was significantly down-regulated by IHC experiment(p<0.05);4.The MAPK3 gene knockout of ESCC with the CRISPR/Cas9 gene editing technology,and the proliferation of ESCC cells after gene knockout had no effect contrast with the control group(p<0.05);5.Organoid culture after MAPK3 gene knockout,morphological changes were observed,that is,synapses appeared and more adherent to walls were not affected in organoid proliferation(p<0.05);6.trans-well experiment suggested that MAPK3 gene knockout significantly promote the invasion and migration of ESCC;7.The ESCC transplantation model experiment indicated that the mice of MAPK3 gene knockout showed obvious invasion and metastasis,with metastasis in lung,kidney,lymph nodes and other places,and the pathological results suggested that the degree of differentiation was poor.Conclusion:In this study,the key gene MAPK3 was screened based on proteomics and bioinformatics analysis,and the expression level of MAPK3 gene was significantly down-regulated in ESCC tissues.Cell,organoid and mouse tumor transplantation experiments confirmed that MAPK3 gene knockout promoted the invasion and metastasis of ESCC.