Effects And Mechanism Of The Orphan GPR50 Receptor On Rat Liver Cells BRL-3a And Hepatoma Cells CBRH-7919

G-protein coupled receptors（GPCRs）,one of the largest family of human membrane proteins,plays an important role in signaling pathways.Due to the high specificity of their ligands and intracellular signaling pathways,GPCRs have larger advantage in drug development.GPR50 is an orphan GPCR located on X chromosome Xq28.Studies had shown that GPR50 may play an important role in lipid metabolism,energy metabolism and breast cancer.However,little research has been done on its role in liver.Considering the advantages of GPCRs in drug screening,studying the role and mechanism of GPR50in liver cells has important reference significance for drug development of liver diseases.Therefore,this study intends to explore the effects and mechanism of GPR50 on hepatocytes（hepatoma cells）in vitro.In our study,rat hepatocytes line BRL-3A and hepatoma cell line CBRH-7919 were used as experimental materials.Firstly,the expression of GPR50 in both cells was detected by qRT-PCR,Western Blot and immunofluorescence.It was found that the expression of GPR50 in CBRH-7919 cells was significantly higher than that in BRL-3A cells,suggesting that GPR50 may play an important role in liver cancer.Then we investigated the effects of GPR50 on the cell viability,cell proliferation,cell cycle and autophagy of the two cells by gene addition or interference with endogenous GPR50 using MTT,EdU,flow cytometry,soft agar assay,wound-healing assay and MDC staining.It was found that the siRNA interference results were poor,probably due to the low background content of GPR50 in the two cells.Fortunately,we successfully obtained GPR50 overexpressing stable cell lines by lentiviral-mediated methods.And the correlation test results showed that overexpression of GPR50 inhibited the proliferation of BRL-3A cells,while promoted the proliferation and migration of CBRH-7919 cells,moreover,promoted autophagy of both cells.To further explore the molecular mechanism of GPR50 regulating BRL-3A cell proliferation,iTRAQ^TM detection revealed significant differences in the expression of 50 proteins in the GPR50overexpression group（3A-GPR50）and the control group（3A-PCDH）.Among them,21 proteins were significantly up-regulated and 29 down-regulated.GO and KEGG enrichment analysis found that the above proteins were mainly involved in physiological activities such as biological regulation,stress response,cell proliferation,metabolic processes and signaling pathways such as PI3K/Akt and cell cycle.The GO function annotation found that 12 proteins were involved in cell proliferation and participated in physiological activities such as cell proliferation regulation,cell cycle regulation,G₁/S phase transformation,and PI3K/Akt signaling pathway.Combined with differential proteomic analysis and the literature,we hypothesized that GPR50 interacting with TβRI induced spontaneous TβRI-dependent Smad signaling pathway to block the G₁/S phase transition of BRL-3A cells by up-regulating P21 and P27expression（which repressed the activity of the cyclin complex CCND1/CDK4/6 and CCNE/CDK2）.Firstly,we verified the interaction between GPR50 and TβRI by immunoprecipitation combined with immunofluorescence.Then,Western Blot detection of TGF-βsignaling pathway and G₁/S phase-related protein expression showed that the results were consistent with our expectations.Furthermore,we also demonstrate the mechanism by adding its signaling pathway inhibitors.