Abstract:

Objective To investigate the effect of ring finger protein 31 （RNF31） on the expression of epidermal growth factor receptor （EGFR）， and to explore the regulation mechanism of RNF31 on EGFR. Methods The interaction between RNF31 and EGFR proteins was validated by co-immunoprecipitation （Co-IP） method.The HEK293T cells were divided into empty vector group（transfcted with pcDNA3.1 emptey vector）and RNF31 group（transfected with RNF31-Flag plasmid）. The expression levels of EGFR mRNA and protein in the cells were detected by real-time fluorescence quantitative PCR （RT-qPCR） and Western blotting methods.After mutation of the ubiquitin ligase function of RNF31 （RNF31^C699/702S）and deubiquitinase binding site （RNF31^{N84A Y93A}），Western blotting method was used to detect the expression levels of EGFR protein in the cells in various groups.The HEK293T cells were divided into empty vector group，RNF31 group（transfected RNF31-Flag plasmid），RNF31+EGFR inhibitor Ggfitinib（Gefitinib） group and RNF31+EGFR inhibitor Erlotinib（Erlotinib） group； the expression levels of EGFR and its downstream sigaling pathway proteins in the cells in various groups were detected by Western blotting method. Results The Co-IP method detection results confirmed that the RNF31 was interacted with EGFR. Compared with empty vector group， the expression level of EGFR mRNA in the cells in RNF31 group was significantly decreased （P<0.05）. Compared with empty vector group， the expression levels of EGFR protein in the cells in RNF31^C699/702S group and wild-type RNF31 group were significantly increased（P<0.05）. Compared with RNF31 group，the expression level of EGFR protein in the cells in transfection RNF31^{N84A Y93A} group was decreased （P<0.05）. Compared with empty vector group，the expression levels of EGFR and nuclear transcription factor-κB（NF-κB）， phosphorylated signal transducer and activiator of transcription 3（p-STAT3），mitogen-activated protein kinase（MAPK） and phosporylated MAPK（p-MAPK） proteins in its downstream signling pathways in the cells in RNF31 group were significantly increased（P<0.05）；the expression levels of protein kinase B（Akt），phosphorylated Akt（p-Akt），STAT3 and MAPK proteins in the cells in RNF31+Gefitinib group and RNF31+Erlotinib group were significantly decreased（P<0.05）. Conclusion RNF31 exerts deubiquitinating through the deubiquitinase enzyme-binding struatural domain， reduces the ubiquitination level of the EGFR protein molecule surface， and slows down the degradation rate of EGFR protein， while highly expressed EGFR can activate the expressions of its downstream proteins related to cell proliferation and division.

Key words: Ring finger protein 31, Epidermal growth factor receptor, Ubiquitination, Protein interaction, Deubiquitinase