SYVN1 Ubiquitinates FoxO1 To Induce P-catenin Nuclear Translocation,PD-LI-mediated Metastasis,and Immune Evasion Of Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma(HCC)is a malignant tumor that poses a serious threat to human health,and its morbidity and mortality rates are increasing every year.There is a lack of effective treatment for advanced HCC,so it is urgent to identify the pathogenesis of HCC and find new therapeutic targets.Recent studies have revealed that HCC cells suppressed the activation of tumor killer cells by overexpression of programmed death ligand 1(PD-L1),thus evading immune system surveillance and maintaining their malignant proliferation and metastasis.However,the molecular mechanisms underlying the high expression of PD-L1 in HCC cells are not fully understood.Previous studies have confirmed that Synoviolin(SYVN1)was a key protein that promoted the development of HCC,but whether it was involved in regulating the expression of PD-L1 in HCC cells and influencing the immune escape of HCC were unknown.MethodsThe UbiBrowser database was used to predict the ubiquitinated substrates of SYVN1.Western blot(WB)and reverse-transcription quantitative PCR(RT-qPCR)were used to detect the expressions of SYVN1 and forkhead box protein O1(FoxO1)in HCC tissues and cells.After knockdown of SYVN1,or overexpression of FoxO1,the expression of PD-L1 in HCC cells was detected by WB and immunofluorescence staining,the proliferation was assessed by CCK-8 assay and clone formation assay,and the migration and invasion ability of HCC cells were detected by cell scratch assay and Transwell migration/invasion assay.HCC cells were co-cultured with human peripheral blood mononuclear cells(PBMCs),treated with or without PD-L1 neutralizing antibodies,and the ratio of CD8+and CD4+T cells was measured using flow cytometry,the content of interferon-y(IFN-γ)in the co-culture supernatant was determined by enzyme-linked immunesorbent assay(ELISA).Co-immunoprecipitation(Co-IP)was used to clarify the binding relationship between SYVN1 and FoxO1 in HCC cells and the ubiquitinated degradation of FoxO1 by SYVN1.Chromatin immunoprecipitation(ChIP)and dual-luciferase reporter assay were used to clarify the binding of FoxO1 and β-catenin to the PD-L1 promoter,and WB was used to assess the regulatory role of FoxO1 and β-catenin on PD-L1 expression.SYVN1 was overexpressed accompanied by overexpression of FoxO1,WB and PBMCs co-culture experiments were performed to assess the regulation of immune escape,proliferation and metastasis of HCC cells by SYVN1 through FoxO1.A subcutaneous xenograft model of HCC cells in NOD/SCID mice and BALB/c nude mice,besides,HCC cells were injected into BALB/c nude mice via the tail vein.The regulatory effects of SYVN1 on HCC immune escape and metastasis were assessed by HE staining and immunohistochemical staining.Results(1)SYVN1 expression was significantly up-regulated,while FoxO1 expression was significantly down-regulated in HCC tissues and cells.(2)After SYVN1 knockdown,the expression of PD-L1 in HCC cells was significantly decreased,the proportion of CD3+CD8+cells was up-regulated,while the proportion of CD3+CD4+cells was down-regulated in PBMCs co-cultured with HCC cells,and the concentration of IFN-y in the coculture supernatant was increased.(3)The proliferation,migration and invasion abilities of HCC cells were significantly reduced after SYVN1 knockdown.(4)Overexpression of FoxO1 decreased the expression of PD-L1 in HCC cells,increased the proportion of CD3+CD8+cells and decreased the proportion of CD3+CD4+cells in PBMCs co-cultured with HCC cells,and increased the content of IFN-y in the coculture supernatant.(5)The proliferation,migration and invasion abilities of HCC cells were significantly decreased after overexpression of FoxO1.(6)SYVN1 directly bound to FoxO1.(7)FoxO1 expression was up-regulated by MG132 in HCC cells.(8)After SYVN1 knockdown,FoxO1 protein level was significantly increased,the half-life of FoxO1 was prolonged,and the ubiquitination level of FoxO1 was significantly decreased in HCC cells.(9)FoxO1 and β-catenin could directly bind to the PD-L1 promoter in HCC cells.Overexpression of FoxO1 reduced the expression of β-catenin in HCC cells,and knockdown of β-catenin reduced the expression of PD-L1 in HCC cells.(10)SYVN1 promotes PD-L1 expression,immune escape,proliferation and metastasis of HCC cells by inhibiting FoxO1.ConclusionSYVN1 promotes immune escape and metastasis of HCC by targeting FoxO1/β-catenin/PD-L1 axis.