The Role And Mechanism Of Lenvatinib Regulating PD-L1 In The Progression Of Hepatocellular Carcinoma

Background:Liver cancer is a common malignant digestive tract tumor in clinical practice,among which hepatocellular carcinoma(HCC)accounts for about 90%of primary hepatic carcinoma.Due to insidious onset,most HCC patients are diagnosed at an advanced stage and can only choose drug therapy.Lenvatinib is the first-line treatment for patients with advanced liver cancer.Studies have shown that lenvatinib can inhibit the expression of programmed cell death protein-ligand 1(PD-L1)in hepatoma cells.However,the effects of lenvatinib on the growth and other biological behaviors of hepatocellular carcinoma cells after regulating PD-L1 expression and the possible mechanisms remain unclear.This paper focuses on the specific action and mechanism of the regulation of lenvatinib on PD-Ll on hepatoma cells,providing a new perspective for the anti-tumor effect of lenvatinib.Objective:1.To investigate the regulatory effect of lenvatinib on the expression of PD-L1 in hepatoma cells;2.To explore the effects of lenvatinib on the growth,migration,invasion and angiogenesis of hepatocellular carcinoma cells by regulating PD-L1,and clarify its possible mechanism of action.This study aims to provide new strategies and scientific basis for the treatment of patients with advanced HCC through the above studies.Methods:1.To investigate the appropriate concentration of interferon gamma(IFNγ)induced up-regulate of PD-L1 in MHCC-97H and Huh7 cells;2.The stable underexpression and overexpression of PD-L1 liver cancer cell lines MHCC-97H and Huh7 were constructed;3.Western blot assay was used to detect the regulatory effect of lenvatinib on the expression of PD-L1 in MHCC-97H and Huh7 cells;4.The effect of lenvatinib on the proliferation ability of MHCC-97H and Huh7 cells was detected by CCK8 assay;5.Transwell experiment was used to investigate the effects of lenvatinib on migration and invasion ability of MHCC-97H and Huh7 cells;6.Determine the influence of lenvatinib on soluble PD-L1(sPD-L1)and VEGFA secretion of liver cancer cells by ELISA experiment;7.Tube formation experiment was used to investigate the effect of lenvatinib on HUVEC cell angiogenesis:8.The expression of important signal proteins in PI3K/AKT signal transduction pathway was analyzed by Western blot assay:9.The mouse model of subcutaneous tumor bearing with MHCC-97H cells was constructed to investigate the effect of lenvatinib on the progression of hepatocellular carcinoma by regulating the expression of PD-L1 in vivo.Results:1.Western blot assay confirmed that 50 ng/ml IFNy was the appropriate concentration to induce the up-regulation of PD-L1 in MHCC-97H and Huh7 cells(P<0.01,P<0.05):2.Stable liver cancer cell lines MHCC-97H1ow(P<0.05),Huh7low(P<0.001).MHCC-97Hhigh(P<0.001).Huh7high(P<0.05)with low and high expression of PD-L1 were successfully constructed;3.Western blot showed that the expression of PD-L1 in MHCC-97H and Huh7 cells could be down-regulated by lenvatinib(P<0.05).and the inhibition was concentration-gradient dependent.4.CCK8 experiment showed that lenvatinib inhibited the proliferation of hepatocellular carcinoma cells by down-regulating the expression of PD-L1;5.Transwell experiment found that lenvatinib could reduce the migration and invasion ability of hepatocellular carcinoma cells by down-regulating the expression of PD-L1;6.Tube formation experiment showed that lenvatinib inhibited the angiogenesis of liver cancer by down-regulating the expression level of PD-L1:7.The ELISA results showed that lenvatinib could reduce the secretion of sPD-L1 and VEGFA by hepatocellular carcinoma cells.8.Western blot showed that lenvatinib inhibited PI3K/AKT signaling pathway of hepatocellular carcinoma cells by down-regulating the expression of PD-L1;9.In vivo experiments showed that lenvatinib could inhibit the growth of subcutaneous tumors in mice by down-regulating the expression of PD-L1(P<0.01).Conclusions:1.Lenvatinib can inhibit the expression level of PD-L1 in hepatoma cells.2.Lenvatinib inhibits PI3K/AKT signaling pathway by down-regulating the expression of PD-L1 in liver cancer cells,thereby suppressing the proliferation,migration,invasion and angiogenesis of liver cancer cells.