C-Met Regulates The Expression And Mechanism Of PD-L1 In Hepatocellular Carcinoma

Background﹠Aims:Hepatocellular carcinoma（HCC）has attracted widespread attention as the third leading cause of death worldwide.Because its onset is relatively insidious,many patients have already reached the middle and late stages of the disease once they are discovered.At present,traditional treatment methods such as surgical resection have little effect on the treatment of advanced hepatocellular carcinoma.Chemotherapy drugs such as Sorafenib,which were first approved by the FDA for the treatment of hepatocellular carcinoma,are also limited by their drug resistance.In recent years,many researchers have devoted a lot of time and energy to the problem of drug resistance of hepatocellular carcinoma.Studies have shown that PD-L1 is usually overexpressed in drug-resistant tumors,and its mediated immune escape may be related to tumor drug resistance.However,the controlled mechanism of PD-L1 in hepatocellular carcinoma remains unclear.This study aimed to elucidate the effect of PD-L1 in hepatocellular carcinoma and its controlled mechanism.Methods:Huh-7^PD-L1+,Huh-7^SRPD-L1-,Huh-7^c-Met+,Huh-7^SRc-Met-,Huh-7^SRp65-and Huh-7^SRp65-c-Met-were successfully constructed by transfecting lentivirus.The protein and m RNA levels of PD-L1 in each cell group were evaluated by Western blot and RT-PCR.Immunochemical methods and indirect immunofluorescence experiments were used to characterize and localize PD-L1 and c-Met in each cell group.The CC-K8 method was used to evaluate the toxicity and cell viability of different concentrations of sorafenib to each cell group.JC-1 method was used to detect the difference of apoptosis in different concentrations of sorafenib in each cell group.Differences in proliferative capacity between cell groups were assessed using Ed U and clonogenic assays.Transwell and scratch healing assays were used to evaluate the migration and invasion abilities of each cell group.The regulation mechanism of c-Met on PD-L1 was verified in vivo by the tumor volume in nude mice.Differences in PD-L1 protein levels between groups were measured by tissue immunohistochemical staining and western blotting.Results:c-Met and PD-L1 were abnormally highly expressed in Huh-7^SR,while their expression levels were lower in Huh-7.Compared with Huh-7,PD-L1 expression was up-regulated in Huh-7^c-Met+cell line.Compared with Huh-7^SR,PD-L1 expression level was decreased in Huh-7^SRc-Met-cell line.When exploring the biological function of PD-L1,we found that in the Huh-7^c-Met+cell line with high expression of c-Met,the proliferation ability and migration and invasion ability were increased by up-regulating the proliferation molecule P70S6K and the migration molecule MMP2.However,in Huh-7^SRc-Met-cell line,the proliferation ability,migration and invasion ability were down-regulated.In the subsequent study on the mechanism of PD-L1 control,we found that c-Met activates the MAPK signaling pathway and the downstream NF-κBp65 transcription factor,interacts with the proximal region of the PD-L1 promoter,and promotes PD-L1 expression.In conclusion,at the cellular level,PD-L1 was down-regulated to varying degrees in both Huh-7^SRc-Met-and Huh-7^SRp65-cell lines compared with Huh-7^SR.In vivo in nude mice,compared with Huh-7^SR,the tumor volume of Huh-7^SRc-Met-,Huh-7^SRp65-and Huh-7^SRp65-c-Met-tumor-bearing mice was significantly down-regulated,and Huh-7^SRp65-c-Met-had the smallest tumor volume.And by inhibiting the activation of the MAPK signaling pathway and then inhibiting the NF-κBp65transcription factor,thereby regulating the expression of PD-L1.It was further demonstrated in vivo that c-Met regulates the expression of PD-L1 through the MAPK/NF-κBp65 signaling pathway.Conclusion:PD-L1 is highly expressed in Huh-7^SR cell line.Furthermore,c-Met was positively correlated with PD-L1 expression levels.c-Met promotes the progression of HCC by regulating the transcription of PD-L1 through the MAPK/NF-κBp65 pathway.c-Met and PD-L1 are expected to be potential targets for the treatment of HCC.Figure[27]Table[0]Reference[50]...