Studies On The Mechanism Of SOX9 Regulating Metastasis Of Hepatocellular Carcinoma

Hepatocellular carcinomas(HCC),with its low survival rates and high postoperative recurrence rates,are a major killer of human beings today.However,the clinical treatment and basic research of HCC have not yet made breakthrough.The development of hepatocellular carcinoma is a long and complex process,gene mutation and tumor microenvironment changes are involved and play an important role.Clinical data statistics found more than 80%of HCCs develop in fibrotic or cirrhotic livers,so the role of liver fibrosis/cirrhotic in the occurrence and metastasis of HCCs has received increasing attention.Hepatic stellate cell(HSC)activation and synthesis of large amounts of extracellular matrix protein is the key to liver fibrosis.Whether hepatoma cell can interact with HSCs to create a microenvironment suitable for tumor growth and metastasis is worth discussing.SOX9,a SRY-related HMG box transcription factor,plays a key role in development.Furthermore,SOX9 is reported to be closely related to the development of a variety of diseases,including tumors,via various mechanisms.Data exploring in Oncomine data platform showed that the expression level of SOX9 in hepatocellular carcinoma was significantly higher than that in normal liver tissue.By comparing the recurrence rate and the expression level of SOX9 in different BCLC stages,we found that the expression level of SOX9 was closely related to the poor prognosis of hepatocellular carcinoma.We then determined the expression of SOX9 in hepatocellular carcinoma cells with different metastatic ability.The results also showed that SOX9 increased in the hepatocellular carcinoma cells with high metastatic ability.In order to further investigate the role of SOX9 in hepatocellular carcinoma metastasis,we constructed the lentivirus that can express SOX9,transfected the low metastatic hepatocellular carcinoma cells SMMC-7721 and obtained SMMC-7721-LV-Sox9 cell that can stably express SOX9.In vitro,we found that overexpression of SOX9 can promote the migration and proliferation of hepatocarcinoma cells,but failed to affect the expression of epithelial-mesenchymal transition(EMT)-related genes,indicating that the role of SOX9 in promoting hepatocarcinoma cell metastasis may be independent of the EMT process.The orthotopic tumor metastasis model in nude mice results also showed that SOX9 overexpression can significantly enhance the liver metastasis and lung metastasis,and promote tumor growth.Simultaneously,we stably knocked down SOX9 in high SOX9-expressing HCC-LM3 cells by transfecting lentivirus carrying SOX9 small hairpin RNA(shRNA).Decrease of SOX9 could inhibit the migration and proliferation of HCC cells,but did not affect the related genes expression of EMT.In vivo results showed that knockdown of SOX9 significantly inhibit the intrahepatic metastasis and lung metastasis of hepatoma cells and inhibit the growth of tumor.Meelis discovered Sox9 could bind to the transcriptional regulatory region of INHBB by ChIP-seq method in the process of hair follicle development in mice.Activin B,consisting of two subunits of Inhibin?B encoded by the INHBB,is one of the TGF-? family members.Western blot results showed that SOX9 increases the expression of INHBB,enhancing the phosphorylation of Smad3 in hepatoma cells and HCC tumors.It turned out that SOX9 may upregulate INHBB and enhance the Activin/Smad signal transduction of hepatoma cells by autocrine.Above conclusion was also confirmed by knockdown of SOX9 in vitro and in vivo.In addition,histological examination and immunohistochemical staining revealed that HSCs activation,liver fibrosis degree and the extent of inflammation significantly increased in the liver tissue adjacent to the carcinoma overexpressing SOX9.Whereas,knockdown SOX9 exhibited converse results.These results suggest that SOX9 upregulates the expression of INHBB in hepatoma cells,which may promote hepatic fibrosis by activating HSCs through paracrine to enhance the metastasis of hepatoma cells.Moreover,ActivinB can activate the human HSC LX-2 and increase the expression of collagen I.Finally,we searched the data in Oncomine,TCGA and GTEx and found that the expression of INHBB in the clinical samples of hepatocellular carcinoma was up-regulated compared with normal liver tissue,and was positively correlated with SOX9.The prognosis was poor when SOX9 and INHBB were both upregulated.The expression of SOX9 and INHBB was also positively correlated with ?-SMA which further indicated that SOX9 induced INHBB expression was correlated with HSC activation and hepatic fibrosis.In conclusion,we found the SOX9 can activate the Activin/Smad signaling in hepatoma cells and HSCs through upregulating INHBB,and thus promote hepatic fibrosis and hepatoma metastasis.Our results extend the knowledge of Sox9 and the understanding of the cross talk between hepatoma cells and HSCs during HCC growth and metastasis,suggesting that it could be an effective target for HCC therapy.