Molecular Mechanism Of Ouabain Regulating PD-L1 In Lung Cancer Cell

The formation and development of cancer is a very complex physiological and biochemical process.In cancer patients,the immune system is no longer able to recognize abnormal tumor cells because tumor cells use immune checkpoints to avoid detection by the immune system.Many monoclonal antibody drugs with good specificity have been developed for tumor cells to escape immune surveillance.PD-L1 and PD-1 are proteins on the plasma membrane.PD-1,also known as CD279,is I class transmembrane receptors,mainly expressed in T cells,B cells,monocytes,natural killer cells and dendritic cells surfaces.For PD-1,two ligands have been found:PD-L1 and PD-L2.PD-L1 is expressed in both hematopoietic and non-hematopoietic cells.Research has found a wide variety of tumor cells are found in the cell surface PD-L1 expression,such as melanoma,lung cancer,breast cancer,malignant glioma,ovarian cancer and bladder cancer,and when the PD-L1 combined with PD-1,it inhibits lymphocyte proliferation and hinder the release of cytokines and cell toxicity,leads to tumor-specific T cells apoptosis.Tumor cells can use PD-L1/PD-1 signaling pathway to achieve the immune escape.Currently,breaking the immunosuppressive signal transduction pathway is the research center of tumor immunotherapy.Various monoclonal antibody drugs targeting the PD-L1/PD-1 signaling pathway are now widely used in clinical practice,and the overall therapeutic effect is about 30%-40%.Some patients also have strong side effects and miss targets in the treatment process.This topic in human non-small cell lung cancer cells as the main research target,adopts theRT-PCR,plasmid overexpressing,siRNA interference,protein immunoblot,protein immune coprecipitation and immunofluorescence confocal experimental methods and technologies.Our experimental results showed that1.Ouabain,the natural small-molecule drug with the most obvious down-regulation of pd-11,was screened in H157,we also confirmed it in H1792,A549,H460 and H1299,and the results indicated that the level of PD-L1 in tumor cells decreased significantly with the increase of ouabain treatment concentration.2.Ouabain mediates ER Stress in tumor cells,and down-regulation of pd-11 depends on the proteasome pathway rather than the lysosomal pathway.3.Overexpression and knockdown of RNF19B in H1792,A549 and H1299 cells confirmed that RNF19B was a new ubiquitin-ligase of PD-L1.4.Ouabain was added to H1299 and A549 and overexpressed RNF19B and two forms of PD-L1.The results showed that RNF19B could enhance the ubiquitination of PD-L1.RNF19B has a strong affinity and ubiquitination level for non-glycosylated PD-L1.5.The overexpression of the segment of PD-L1 and the segment of RNF19B in HEK293FT showed that RNF19B bound to the cytoplasmic segment of PD-L1 through the structural region of 252-732AA,Meanwhile,ubiquitin molecules mutated at different sites were used to reveal the ubiquitination form of RNF19B to PD-L1.6.The results of immunoprecipitation showed that RNF19B interacts with USP22,affecting the intracellular stability of PD-L1.Conclusion:In non-small cell lung cancer using ouabain treatment,it can cause cancer cells to produce calcium homeostasis disorders and it will induce endoplasmic reticulum stress.The synthesis of PD-L1 under the ER.Stress is hindered.PD-L1 is recognized and ubiquitinated by endoplasmic reticulum ubiquitin ligase RNF19B Ubiquitinated PD-L1 was sent to 26S proteasome.At the same time,we also found that RNF19B can affect the binding of the deubiquitination enzyme USP22 to PD-L1 and eventually reduce the stability of PD-L1.