EFEMP2 Induces PD-L1 Expression Via EGFR/ERK1/2/c-Jun Signalling In Ovarian Cancer

BackgroundEarly detection is crucial to the treatment and survival of cancer patients,and the discovery of cancer biomarkers is a revolution in cancer screening.They have great prospects in the treatment roadmap and can reduce costs and cancer-related mortality and morbidity.However,despite great efforts in this area,only a few cancer biomarkers have been approved by the FDA.Symptoms are not obvious,leading to high spread of the disease in the later stage,lack of appropriate screening and chemotherapy resistance,all of which lead to poor diagnosis and failure of treatment.In the past ten years,the pursuit of cancer metabolism biomarkers has been very strong,but its promotion to the clinic has not been accepted,mainly because of the lack of a clear and proven mechanism link with cancer metabolism.Our study used lentiviral transfection to interfere with or overexpress the expression of EFEMP2 to explore the relationship between EFEMP2 and the invasion and migration of ovarian cancer cells,and then use proteomics and other methods to explore pathways that may affect the invasion and migration of ovarian cancer cells.To better understand the molecular mechanisms of ovarian cancer occurrence and development.Research purposesThe regulatory relationship between EFEMP2 and PD-L1 is unclear.Our study explored the regulatory relationship between EFEMP2 knockout and PD-L1 expression in ovarian cancer,and further explored its specific signaling pathways to find biomarkers for ovarian cancer.Research method1.Use Kaplan-Meier Plotter and Oncomine public databases to analyze the relationship between EFEMP2 and ovarian cancer;2.Quantitative analysis of EFEMP2 mRNA in four ovarian cancer cells ES-2.SKOV-3,CAOV-3 and OVCAR-3 by RT-qPCR:3.Use immunocytochemistry and western blotting to compare the expression differences of EFEMP2 protein in four different human ovarian cancer cells;4.The results of the Transwell chamber test reflect the differences in migration and invasion of the cell lines;5.Transfection with lentivirus,and verify its transfection efficiency;6.Plate clone formation test can reflect cell population dependence and cloning potential;flow cytometry reflects changes in the ability of cell apoptosis;7.The expression of EMT-related genes in the cells before and after transfection was detected by western blotting;8.Establish a tumor transplantation model in nude mice,and regularly record tumor growth and size;9.Use proteomics to analyze the differential genes in ovarian cancer cells before and after knocking down EFEMP2.and perform KEGG pathway enrichment analysis on the differential genes to find out the protein pathways related to EFEMP2;10.Detect the expression of EGFR/ERK1/2/c-Jun pathway and PD-L1 in ovarian cancer cells by western blotting;11.Use immunoprecipitation to further detect the binding effect of EFEMP2 with downstream proteins.Research result1.Kaplan-Meier Plotter and Oncomine public databases show that patients with low EFEMP2 expression have a higher survival rate,and the expression of EFEMP2 in ovarian cancer is significantly higher than that in normal ovaries;2.The RNA and protein expressions of the four cell lines are ES-2,SKOV-3,CAOV-3,and OVCAR-3 from high to bottom,respectively.Ovarian cancer cells with high expression of EFEMP2 have stronger migration and invasion ability;3.The transfection efficiency is greater than 80%,the migration and invasion ability of ES-2 shRNA was significantly decreased,while OVCAR-3 EX was significantly increased;4.The clone-forming ability and tumorigenicity of the cell line with high EFEMP2 expression is stronger,and its apoptotic ability is weaker,while the opposite was true in the cells with low expression;5.Western blotting results showed that the EMT related genes in the ES-2 shRNA group generally decreased,and the OVCAR-3 EX group generally increased;6.The results of the protein chip show that EGFR and c-Jun are differential genes.In the enrichment analysis of the KEGG pathway,the PD-L1 checkpoint is significantly enriched,and the protein expression of the EGFR/ERK1/2/c-Jun pathway is consistent with the expression of EFEMP2;7.Co-immunoprecipitation results show that EFEMP2 binds to EGFR.Conclusion and significanceThis is the first time to clarify the regulation mechanism of EFEMP2 induced PD-L1 in ovarian cancer,which can be mediated by the activation of EGFR/ERK1/2/c-Jun.In two ovarian cancer cell lines with different expression levels of EFEMP2,knockdown or overexpression of EFEMP2 proved that EFEMP2 can regulate the expression of PD-L1 in ovarian cancer through the EGFR/ERK1/2/c-Jun pathway,which provides new markers of ovarian treatment.Based on the characteristics of EFEMP2 in other tumors and our analysis in ovarian cancer,we believe that EFEMP2 can be used as a marker for ovarian cancer in the future.