UBE2S Promotes Malignant Progression Of Non-small Cell Lung Cancer By Ubiquitination Degradation Of RPL26

Non small cell lung cancer(NSCLC)is the type with the highest proportion of lung cancer.It is usually diagnosed in the late stage.In view of the poor prognosis and low survival rate of patients with advanced NSCLC,a variety of targeted drugs have been applied to the clinical therapy of patients with advanced NSCLC.However,most clinical patients will develop resistance to these drugs.Therefore,the search for new therapeutic targets is of great significance for the clinical therapy of NSCLC.Generally,ubiquitin binding enzyme E2 mainly plays the function of transmitting ubiquitin molecules,but some E2 enzymes can also interact with substrates and directly ubiquitinate substrates without the participation of E3 ligase.At present,some small molecule inhibitors for E2 enzyme,such as CC0651 and leucettamol A,have achieved preliminary results in tumor therapy,indicating that E2 enzyme can be used as a candidate target for tumor therapy.In present study,the E2 enzyme UBE2 S was screened to be highly expressed in NSCLC,and its biological function,new substrate and ubiquitination mechanism in NSCLC were explored.(1)UBE2S promotes the proliferation,migration and stemness of NSCLCIn TCGA database,250 genes with up-regulated expression in NSCLC were selected and intersected with 37 members of E2 enzyme family.Then,combined with GEPIA database analysis,the highly expressed E2 enzyme in NSCLC was screened.Using normal lung epithelial cell line BEAS-2B,NSCLC cell lines(A549,H1299,H520,H460)and lung cancer tissue array as experimental materials,RT-q PCR,WB and immunohistochemistry experiments were applied to identify UBE2 S as research object in NSCLC.GEPIA database analysis showed that NSCLC patients with high UBE2 S expression had a low overall survival(OS).After interference / overexpression of UBE2 S in A549 and H1299 cell lines,respectively,it was determined that UBE2 S promoted the proliferation,migration and stemness of NSCLC cells by CCK8,wound scratch and spheroidization experiments.(2)Substrate identification and regulatory mechanism of UBE2 S in NSCLCThe proteins interacting with UBE2 S in NSCLC cells were screened and identified by co-immunoprecipitation(IP)combined with mass spectrometry(MS)technology.GO and KEGG analysis showed that most of these proteins belong to ribosomal proteins and their molecular functions and biological processes are related to ribosomes.In A549 and H1299 cells with UBE2 S interference or overexpression,the top five interacting proteins in MS data were detected by WB.It was found that only RPL26 was regulated by UBE2 S at the protein level,and the expression of RPL26 in NSCLC cell lines was lower than that in normal lung epithelial cell line.In A549 cells,the interaction between UBE2 S and RPL26 was confirmed by co-IP assay,and their co-localization in nucleus and cytoplasm was shown by immunofluorescence staining.The half-life of protein experiment indicated that UBE2 S could reduce the stability of RPL26 protein and shorten its half-life.Treatment with proteasome inhibitors also reversed the negative regulation of UBE2 S on RPL26.Finally,the endogenous ubiquitination IP experiment confirmed that UBE2 S could enhance the ubiquitination modification level of RPL26.The above data shows that UBE2 S degrades RPL26 through ubiquitin-proteasome pathway.(3)UBE2S plays a carcinogenic role through ubiquitin degradation of RPL26Functional rescue experiment was used to explore whether the tumor-promoting effect of UBE2 S was related to the degradation of RPL26 by ubiquitin.Empty vector pc DNA3.1 and p EGFP-C3 were co-transfected into A549 and H1299 cells as control group,oe-UBE2S(p EGFP-C3+pc DNA3.1-3x HA-UBE2S)group,oe-RPL26(pc DNA3.1+p EGFP-RPL26)group and oe-UBE2S/RPL26(pc DNA3.1-3x HA-UBE2S+ p EGFP-RPL26)co-transfection group were used as experimental groups,and then CCK8,scratch and spheroidization formation experiments were performed.The results showed that RPL26 could reverse the promoting effect of UBE2 S on the proliferation,migration and stemness of NSCLC to a certain degree.In addition,the relationship between UBE2 S and P53,P73,c-Myc proteins was also studied.WB results showed that UBE2 S could down-regulate the expression of P53 protein in A549 cells(P53 wild type).In A549 and H1299(P53 null)cells,UBE2 S had no significant effect on the expression of P73 protein,but it can up-regulate the expression of c-Myc.After co-expression of UBE2 S and RPL26 in A549 and H1299 cells,it was observed that the positive regulation of c-Myc by UBE2 S was inhibited by the overexpression of RPL26,indicating that UBE2 S could play a carcinogenic role through the RPL26-c-Myc pathway in NSCLC.In summary,UBE2 S was screened as the highly expressed E2 enzyme in NSCLC,and ribosomal protein RPL26 was identified as one of its substrates.It is confirmed that UBE2 S could degrade RPL26 through ubiquitin proteasome pathway,thus enhancing the expression of c-Myc,promoting the proliferation and stemness of NSCLC and playing a carcinogenic function.The newly discovered UBE2S/RPL26/c-Myc pathway may provide a new direction for the selection of therapeutic targets for NSCLC and the development of inhibitors.