Roles Of Carcinoma Associated Fibroblasts In Hepatocellular Carcinoma Metastasis And Its Mechanism

Objectives:Hepatocellular Carcinoma(HCC)has a high degree of malignancy in malignant tumors.It is one of the most common malignant tumors of the digestive system in the world.About 2 to 1 million HCC patients die every year.The main cause of poor prognosis is mainly the recurrence and metastasis.The CAF is one of the important components of the TME of HCC and are closely related to the invasion and metastasis of liver cancer.Therefore,the purpose of this study was to investigate the specific mechanisms by which role CAF played in HCC invasive ness and metastasis.Method: 1.Hepatoma tissue samples and paracancerous tissue samples were collected,and the extracted HCC CAF and PTF were isolated and identified.The expression trends of ?-SMA and Vimentin in the two fibroblasts were analyzed,and the migration and proliferation activities of CAF and PTF were compared.2.NOD/SCID mice were used to establish a subcutaneous tumor model to initially verify the effect of CAF and PTF on subcutaneous of hepatoma cells.The effects of CAF and PTF on migration,invasion,and EMT of HCC cells were compared in vitro by using transwell assay,wound healing assay,and Western blotting.3.The protein chip was used to detect differences in secreted proteins of CAF and PTF and Elisa validated the results of the protein chip.RT-PCR was performed on liver cancer tissue samples to analyze the correlation between the secreted factor screened out by the protein chip and the expression of ACTA2 at the mRNA level. We used NOD/SCID mice to establish subcutaneous intratumo ral models,transwell chamber assay,wound healing assay,and Western blotting to verify that the secreted factor impacted in the process of liver cancer development,proliferation, migration,invasion and EMT.4.Hif1? is the most common factor in promoting tumor invasion and metastasis under hypoxic microenvironment.We added CAF-CM,the secreted factor screened out by protein chips,and inhibitors of the secreted factor to hepatoma cells. RT-PCR,Western blotting,and immunofluorescence of frozen sections of subcutaneous tumors were used to verify the secreted factors functioned in the expression of Hif1? in hepatoma cels.5.We established an in vivo model of NOD/SCID mouse subcutaneous tumors. Simultaneously,Transwell chamber assay,wound healing assay,and Western blotting were performed to verify that the secreted factor affect EMT and migration of hepatocellular carcinoma cells by affecting the expression of Hif1? in vitro and in vivo.We used the online tool ProteinAltas to analyze the effect of Hif1? expression on the survival time of HCC patients.Meanwhile,the effect of CAF on ubiquitination of Hif1? under normoxic conditions was verified by CoIP.6.Western blotting were used to verify the effect of Hif1? on the expression of the EMT transcription factor ZEB1 at the protein level.ChIP was then used to verify the effect of Hif1? on the transcription of the downstream target gene ZEB1.We performed RT-PCR and frozen section double-fluorescent staining of liver cancer tissue samples to analyze the correlation between the expression of Hif1? and ZEB1 in liver cancer tissues.Transwell chamber assay,wound healing assay,and Western blotting experiments were used to verify the effect of ZEB1 on EMT, migration and invasion of hepatoma cells.Finally,the effect of ZEB1 expression on the survival time of liver cancer patients was analyzed by the online tool ProteinAltas.Result:1.HE staining and immunohistochemical staining showed that the distribution of activated mesenchymal cells in tumor tissue of HCC patients was significantly wider than that in adjacent tissues.Both CAF and PTF have fibroblast characteristics and are not contaminated by epithelial cells and endothelial cells. Western blot results showed that CAF expressed ?-SMA and Vimentin higher than PTF.CAF has stronger in vitro migration and proliferation than PTF.2.Experiments In vivo showed that CAF can promote the lung metastasis of hepatocellular carcinoma cells.The survival analysis results suggest that CAF can increase the degree of malignancy of liver cancer cells.Increased EMT levels in CAF-CM-incubated HCC cells and more significant migration and invasiveness promoted by CAF.3.We used protein microarray to screen out the chemokine CCL5 with the greatest difference in the expression of secreted factors between CAFs and PTFs.The expression of CCL5 and ACTA2 in the liver cancer tissues showed a positive linear relationship.CCL5 can promote EMT in hepatoma cells and significantly enhance migration and invasion.4.Both in vivo and in vitro experiments have demonstrated that CCL5 can promote the expression of Hif1? in HCC cells.5.The CCL5 secreted by CAF can inhibit the ubiquitin degradation of Hif1?, promote the EMT of hepatocellular carcinoma cells,and increase the migration a nd invasion ability.The online tool survival analysis results suggest that the abnormal increase of Hif1? expression in HCC tissues means tumor progression.6.The experimental results suggest that the activation of CAF-activated Hif1? promotes the transcription of the downstream target gene ZEB1,and the EMT level of hepatoma cells increases at the same time the migration and invasion abilities increase.The survival analysis results suggest that the abnormal increase of ZEB1 expression in HCC tissues means poor prognosis.Conclusion:CAF isolated from human hepatocellular carcinoma can induce Hif1?/ZEB1 expression through the secretion of chemokine CCL5 and promote EMT in hepatoma cels to obtain strong invasion and metastasis capacity.