Study On Post-translational Modification And Correlation Of PD-L1 Protein

Background: The growth and metastasis of non-small cell lung cancer(NSCLC)are closely related to the immune microenvironment.A variety of immune cells and immune molecules are involved in the occurrence and development of tumors.Programmed death receptor-ligand 1(PD-L1)is one of the important immune checkpoint molecules.PD-L1 is expressed on the surface of more than half of NSCLC cells,which leads to poor prognosis by inhibiting T cell function and promoting tumor cell immune escape.The expression of PD-L1 is mainly regulated by transcription,translation,and post-translational modification,of which post-translational modification is the most direct and important regulatory mechanism.In this study,we screened out the two molecules of post-translational modification of PD-L1 protein from two aspects of positive regulation and negative regulation.We explored the mechanism and function of the two molecules in NSCLC by regulating the level of PD-L1,thus providing new methods and strategies for immunotherapy of NSCLC.Among the molecules that positively regulate PD-L1,we found SIRT2.Sirtuin family is a kind of highly conserved deacetylase,which is involved in cell stress response,metabolic process,senescence,apoptosis,and other cellular activities.Among the 7 sirtuin members,only SIRT2 exists mainly in the cytoplasm,which may be co-located with PD-L1.SIRT2 plays an important role in the occurrence and development of tumor.SIRT2 affect the invasion and metastasis of tumor cells by regulating tumor microenvironment.SIRT2 can deacetylate some proteins and change their function and activity.However,the effect of SIRT2 on PD-L1 has not been reported yet.Among the molecules that negatively regulate PD-L1,we found the long noncoding RNA(lnc RNA)LINC02418.Lnc RNA plays a key role in the transcription,translation,and post-translational modification of biological protein molecules,thus affecting the occurrence and development of cancer.Several studies have shown that some lnc RNAs can affect the growth and metastasis of tumor cells by regulating the expression of PD-L1.Several kinds of lnc RNAs that up-regulate PD-L1 have been found to promote tumor immune escape by increasing the expression of PD-L1 or inhibiting PD-L1 degradation,resulting in tumor proliferation,invasion,and migration.There are few studies on lnc RNAs that can reduce the expression of PD-L1.To explore the lnc RNA that negatively regulates the expression of PDL1 may be of far-reaching significance to clinical treatment.Methods: 1.The eukaryotic expression plasmid of PCMV-myc-PD-L1(hereinafter referred to as myc-PD-L1)was constructed and its expression in 293 T cells and A549 cells was verified by western blot.2.The constructed myc-PD-L1 plasmid was transfected into293 T cells and treated with deacetylase inhibitors NAM and TSA,the existence of deacetylation modification of exogenous PD-L1 was verified by co-immunoprecipitation experiment.The existence of deacetylation modification of endogenous PD-L1 was verified by co-immunoprecipitation experiment after treatment of NSCLC cells with NAM and TSA.3.The deacetylase of exogenous PD-L1 was screened by co-immunoprecipitation in 293 T cells,the deacetylase of endogenous PD-L1 was verified by western blot in NSCLC cells,and the co-localization of the deacetylase and PD-L1 was detected by immunofluorescence experiment.4.The expression of PD-L1 and the selected deacetylase in different NSCLC cell lines were compared by western blot,and the suitable cell lines were selected for cell function test.5.To verify that the deacetylase positively regulates the expression of PD-L1.After overexpressing or knocking down the deacetylase according to a specific sequence,the level of PD-L1 was compared by western blot and reverse transcription-polymerase chain reaction RT-PCR).6.Flow cytometry was used to detect the effect of deacetylase on the level of PD-L1 on the surface of NSCLC cells,and flow cytometry was used to detect the effect of deacetylase on the binding strength of PD-L1 to PD-1 on the surface of NSCLC cells.7.Peripheral blood mononuclear cells(PBMC)were segregated from the whole blood of healthy experimental technique workers.PBMC was activated and co-cultured with NSCLC cells transfected with different plasmids for twelve hours.Flow cytometry was used to detect the apoptosis of NSCLC cells.8.Western blot was used to verify the effect of different concentrations of SIRT2 inhibitor sir Real2 on PD-L1 in NSCLC cells.Flow cytometry was used to detect the effect of sir Real2 and PD-L1 monoclonal antibody Atezolizumab on PBMC killing NSCLC cells.9.Bioinformatics technology was used to screen lnc RNAs that negatively regulate PD-L1 and related to the good prognosis of NSCLC.Western blot and RT-PCR were used to verify the effect of lnc RNAs on PD-L1.10.The subcellular localization of the lnc RNA and PD-L1 was detected by fluorescence in situ hybridization.11.Flow cytometry was used to detect the effect of the lnc RNA on the expression of PD-L1 on the surface of NSCLC cells and the binding strength of PD-1,and the effect of the lnc RNA on PBMC killing NSCLC cells was detected by flow cytometry.12.We used western blot,RTPCR and immunoprecipitation to explore the molecular mechanism of down-regulation of PD-L1 level by lnc RNA.13.In order to construct a stable expression cell line,we constructed the lnc RNA-related lentiviral clone and infected the target cells.14.The mouse tumor model was established,and the effect of lnc RNA on tumor tissue in vivo was verified by animal experiments and immunohistochemistry.15.Fluorescence in situ hybridization(FISH)and immunohistochemistry were used to explore the correlation between lnc RNA and PD-L1 in human tissue specimens.16.The effect of the lnc RNA on tumor immune infiltrating cells was analyzed by bioinformatics technique.Results: PCMV-myc-PD-L1 clones were successfully constructed and expressed in293 T cells and A549 cells.When exogenous PD-L1 molecules were transfected into 293 T cells,the acetylation reaction was enhanced by NAM and TSA treatment,and the PD-L1 acetylation reaction was enhanced by NAM treatment in NSCLC cells.In the SIRT family,only SIRT2 interacts with PD-L1.SIRT2 can up-regulate the expression of PD-L1 in NSCLC cells,and SIRT2 and PD-L1 are co-localized in the cytoplasm.In the existing NSCLC cell line A549,the protein levels of PD-L1 and SIRT2 were the lowest,while those of PD-L1 and SIRT2 were the highest in H1703 cells.After overexpression of SIRT2,the level of PD-L1 on the cell surface increased,the binding strength of PD-L1 to PD-1 increased,and the killing effect of immune cells on NSCLC cells was weakened.Sir Real2,an inhibitor of SIRT2,could down-regulate the PD-L1 level of NSCLC cells,and NSCLC cells were more easily killed by PBMC after adding Sir Real2.In the co-culture of PBMC and NSCLC cells,the killing effect of Sir Real2 combined with Atezolizumab was more significant than that of Atezolizumab alone.Three kinds of Lnc RNAs: LINC02418,LINC00339 and LINC00899 were screened out.They were negatively correlated with the level of PD-L1,and the overall survival rate of patients with high expression of Lnc RNAs in NSCLC patients was higher.The protein level showed that only LINC02418 could down-regulate the expression of PD-L1.There is subcellular co-localization of LINC02418 and PD-L1 in the cytoplasm.After knocking down LINC02418,the level of PD-L1 on the surface of NSCLC cells was up-regulated,and the binding strength of PD-L1 to PD-1 was increased.NSCLC cells overexpressing LINC02418 were more likely to be killed by PBMC.After being treated with proteasome inhibitor,the regulatory effect of LINC02418 on PD-L1 was weakened.LINC02418 can enhance the ubiquitin level of PD-L1.LINC02418 could enhance the binding of E3 ligase Trim21 and PD-L1.However,when Trim21 was knocked down,the regulatory effect of LINC02418 on PD-L1 was significantly weakened.When Trim21 was knocked down,the killing effect of PBMC on NSCLC was not affected by LINC02418.In the mouse lung cancer model treated with PD-L1 monoclonal antibody,the tumor growth was the slowest,the tumor volume was the smallest and the proliferation ability was the weakest in the mice overexpressing LINC02418.The high expression of LINC02418 promotes the proliferation and activation of initialized B cells,plasma cells and activated NK cells.Conclusion: The deacetylation modification exists in PD-L1,and SIRT2 is the deacetylase of PD-L1 in NSCLC.SIRT2 is a positive regulatory molecule of PD-L1,which induces immune escape of tumor cells by up-regulating the level of PD-L1.Sir Real2,an inhibitor of SIRT2,promotes the killing of immune cells to NSCLC cells by reducing the expression of PD-L1.There may be a synergistic effect between sir Real2 and Atezolizumab to enhance the killing effect of immune cells.LINC02418 is a negative regulatory molecule of PD-L1 in NSCLC.LINC02418 is a tumor suppressor molecule.LINC02418 degrades PD-L1 protein through Trim21-mediated ubiquitin proteasome pathway and enhances the killing effect of immune cells on tumor cells.LINC02418 may enhance the therapeutic effect of PD-L1 monoclonal antibody.LINC02418 enhances the immune response of the body by activating some immunoreactive cells.SIRT2 and LINC02418 may be new targets for NSCLC immunotherapy.