| Background and objective:Liver cancer is one of the most common malignant tumors of the digestive system in China,with Hepatocellular carcinoma(HCC)being the main type of pathology.Metastasis is a key event in cancer progression and a major barrier to improving long-term survival in advanced HCC.Anoikis resistance is considered a critical step in the initiation and spread of metastasis.Tissue factor(TF),as an exogenous coagulation initiator,can promote a hypercoagulable state in patients and lead to tumor cell metastasis.Secondly,it has been found that the expression of TF in cancer cells is associated with changes in the body’s immune system leading to tumor progression,including adaptive immunity represented by T lymphocytes.Therefore,the aim of this study was to explore the effect of TF on Anoikis resistance in HCC and its mechanism,as well as to investigate whether TF can lead to immune escape of tumor cells and affect the efficacy of immunotherapy,providing a new theoretical basis for the treatment of hepatocellular carcinoma.Methods:(1)The expression level of TF in hepatocellular carcinoma(HCC)and adjacent tissues were detected by immunohistochemical staining and Western Blot techniques,and the relationship between TF expression level and clinicopathologic features of HCC patients was analyzed by a univariate chi-square,as well as the prognostic value of TF expression level in HCC patients.(2)TF protein expression levels in normal liver cells L02 and HCC cell lines MHCC97-H,HCCLM3,SMMC-7721 and Hep3B were detected by q RT-PCR and Western Blot,as well as TF expression in these cell lines suspended for 24h and 48h.stable downregulated TF cell lines were constructed by infecting HCC cells HCCLM3and SMMC-7721 with lentivirus,and a stable overexpressed TF cell line was constructed in MHCC97-H cells.(3)Flow cytometry and Western Blot were utilized for the detection and analysis of the apoptosis of HCC cells in the adherent and suspended states.The invasion and migration ability of hepatocellular carcinoma cells were detected by the transwell and scratch assays.In addition,SCID mice were used to establish a liver metastasis model by tail vein injection of hepatocellular carcinoma cells.(4)The influence of TF on glycolysis pathway was analyzed by detecting lactic acid,ATP and ROS.Key and major glycolysis genes changes in HCC cells modified by TF gene and the effects of lentivirus overexpression of PDK4 on cell apoptosis,lactic acid,ROS and ATP levels in suspension state were detected.Furthermore,the effects of PDK4-mediated glycolysis on TF regulation of anoikis resistance in HCC cells were analyzed.(5)Down-regulated differential protein of the TF gene was analyzed by mass spectrometry,and a regulatory relationship between TF and differential gene was verified by q RT-PCR and Western Blot.The relationship between TF and differential protein was determined by CO-IP and immunofluorescence,and the expression of differential protein was restored.Effects of TF on apoptosis,lactic acid,ROS,and ATP levels were observed.(6)hepa1-6 mouse hepatoma cells were used to establish a liver metastatic model,and the effect of down-regulated TF gene combined with PD-1 monoclonal antibody on liver metastasis was observed.Immunohistochemical techniques were used for the observation of local lymphocyte infiltration in tumor morphologies.After TF gene modification,PD-L1 were detected by Western Blot.Flow cytometry was used to measure CD8a~+T cell levels after co-culture of peripheral blood mononuclear cells with tumor cells.Results:(1)TF m RNA levels were significantly higher in hepatocellular carcinoma tissues than in paraneoplastic tissues;TF was mainly localized in the cell membrane and cytoplasm,and high TF expression was closely associated with envelope integrity,portal vein thrombosis,lymph node metastasis,extrahepatic metastasis and poor prognosis of HCC patients.(2)Compared with L02 in normal hepatocytes,TF was highly expressed in HCCLM3,SMMC-7721 and Hep3B cell lines and was less expressed in MHCC97-H cell line.Compared with the adherent state(att),TF expression level significantly increased in HCC cells HCCLM3,SMMC-7721,Hep3B and MHCC97-H suspended for 48h,suggesting that high expression of TF in HCC cells in suspension may be associated with anoikis resistance in cancer cells;(3)When SMMC-7721 and HCCLM3 cells were suspended,the apoptotic rate and apoptotic protein expression of sh-TF group significantly increased compared with sh-Ctrl group,but the apoptotic rate of the sh-TF group did not have any changes when SMMC-7721 and HCCl M3 cells were suspended.Collectively,compared with the sh-Ctrl group,the invasion and migration of cells in the sh-TF group significantly decreased.Similarly,in MHCC97-H cell line,the apoptosis rate and apoptotic protein expression of OE-TF group significantly decreased in suspension state in comparison with OE-Ctrl group,but there was no significant difference between the two groups in the adherent state.Secondly,compared with the OE-Ctrl group,the invasion and migration ability of cells in the OE-TF group increased significantly.In the liver metastatic tumor model animal experiments,compared with sh-Ctrl group,the number of liver metastases in sh-TF group decreased,suggesting that TF promoted the anoikis resistance,invasion and migration of HCC cells,leading to tumor metastasis.(4)In terms of mechanism,when SMMC-7721,HCCLM3 and MHCC97-H cells were suspended,the levels of lactate and ROS increased,while the levels of intracellular ATP decreased.In SMMC-7721 and HCCLM3 suspension state,compared with sh-Ctrl group,the levels of lactate and ROS in sh-TF group decreased,while the levels of intracellular ATP increased.Similarly,in MHCC97-H suspension state,compared with OE-Ctrl group,lactate and ROS levels in OE-TF group increased,while intracellular ATP levels decreased,suggesting that TF might regulate anoikis resistance in HCC cells through glycolytic level.In order to investigated how TF affected anoikis resistance of hepatocellular carcinoma cells through glycolysis pathway,we examined the 14 glycolytic genes and found that PDK4 m RNA was downregulated more significantly in SMMC-7721 and HCCLM3 cell suspension than in sh-Ctrl group.In MHCC97-H cell line,compared with the OE-Ctrl group,PDK4m RNA was upregulated,and the differential expression of PDK4 was verified at the protein level,and it was surprising to find that in the sh-Ctrl group,compared with the adherent state,the expression level of PDK4 in the three cell lines in suspension state was significantly increased.These results suggested that PDK4 might play a role in TF-mediated anoikis resistance of hepatocellular carcinoma cells.In order to verify the above hypothesis,we found that compared with sh-TF+OE-Ctrl group,sh-TF+OE-PDK4 group could partially reverse the expressions of lactate,ATP and ROS,and reduce the apoptotic rate and the expression of apoptotic proteins.TF regulated anoikis resistance of hepatocellular carcinoma cells through PDK4.(5)The expression of RPS24 was found in both the adherent group and the suspension group by cell mass spectroscopy,and TF positively regulated the expression of RPS24 at the gene and protein levels.Surprisingly,in the sh-Ctrl group,compared with the adherent state,the expression level of RPS24 in cell suspension increased significantly.In addition,TF and RPS24 proteins were found to have a bond,and under a confocal microscopy,we could observe the fluorescence fusion of the two proteins.In order to further investigated the function of RPS24,overexpression of RPS24 could partially reverse the expressions of PDK4,lactate,ROS and ATP,and reduce the apoptosis rate and the expression of apoptotic protein in TF down-regulated cells.These results indicated that TF regulated PDK4-mediated anoikis resistance of hepatoma cells through RPS24.(6)The animal model of liver metastasis was established in C57BL/6 mice.Compared with sh-Ctrl+Ig G group,sh-TF+Ig G group and sh-Ctrl+PD-1 group,the number of liver metastases in sh-TF+PD-1 group was significantly reduced.There was no significant difference in the number of liver metastases among sh-Ctrl+Ig G,sh-TF+Ig G and sh-Ctrl+PD-1 groups.Additionally,the local CD8a~+T lymphocyte infiltration increased in the sh-TF+PD-1 group,while there was no significant difference in the number of CD8a~+T lymphocyte infiltration in the sh-Ctrl+Ig G,sh-TF+Ig G and sh-Ctrl+PD-1 groups.This was associated with down-regulation of TF to regulate glycolysis in hepatocellular carcinoma cells to further inhibit PD-L1expression in cancer cells,while increasing the number of CD8a~+T cells,thus combining with anti-PD-1 monoclonal antibody was able to inhibit immune escape of hepatocellular carcinoma cells and provide efficacy of hepatocellular carcinoma treatment.Conclusion:(1)TF was highly expressed in hepatocellular carcinoma tissues and hepatocellular carcinoma cell lines,and was closely associated with patients’clinical metastatic characteristics and poor prognosis;(2)TF combined with RPS24 regulated anoikis resistance and PD-L1 expression of hepatocellular carcinoma cells and local CD8a~+T cell infiltration through the PDK4-mediated glycolytic pathway.(3)In addition,down-regulation of TF combined with anti-PD-1 monotherapy inhibited hepatocellular carcinoma cell metastasis and increased the efficacy of hepatocellular carcinoma treatment. |
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