Curcumin Inhibits Invasion And Metastasis Of Human Hepatoma Cells Through Bclaf1-mediated Of Wnt/β-catenin Signaling

Background: The early symptoms of primary liver cancer are difficult to detect since the disease develops slowly.As a result,it is difficult to identify,and most patients are diagnosed in the middle or late stage of the disease.Surgical resection is the therapy of choice for liver cancer at the moment,however the treatment of primary liver cancer is still not ideal due to postoperative metastasis and recurrence,as well as tumor medication resistance.Chinese medicine is widely used to prevent and treat cancer,and it is one of the effective treatment for advanced liver cancer.Curcumin is primarily an active element extracted from Zingiberaceae family of plants,and it has low toxicity and adverse effects,and shows a promising future for development and application prospect in the treatment of liver cancer.However,the anti-tumor mechanism of curcumin in the invasion and metastasis of liver cancer is completely unclear.Bcl-2-associated transcription factor 1(Bclaf1)was originally identified as a pro-apoptotic and transcription-associated regulator.The abnormal expression of Bclaf1 is closely associated with various biological processes in hepatocellular carcinoma.The Wnt/β-catenin signaling pathway is highly conserved and plays an important role in the organism.Abnormalities in the cascade response of this pathway can promote cancer stem cell renewal,proliferation and differentiation,leading to the occurrence and development of different liver diseases and hepatocarcinoma.The purpose of this study was to explore the mechanism of curcumin in inhibiting the invasion and metastasis of human hepatocellular carcinoma cells through regulating Wnt/β-catenin pathway by Bclaf1.Objective: This study intends to investigate the inhibitory effect of curcumin on invasion and metastasis of human hepatocellular carcinoma cells HepG2 and SK-Hep-1 through Wnt/β-catenin signaling pathway and the molecular mechanism of Bclaf1 regulation through in vivo and in vitro expriments.Methods: The effects of curcumin(0,10,20,40,60 μM)and 5FU(10 μM)on the proliferation of human hepatocellular carcinoma cells HepG2 and SK-Hep-1 were detected by CCK8 assay.Moreover,the migration ability of curcumin-treated human liver cancer cells in vitro was detected by scratch healing test,and the invasive ability of curcumin-treated human liver cancer cells in vitro was detected by Transwell chamber.Western blot was adopted to detect the expression of Bclaf1,invasion and metastasis and associated proteins of Wnt/β-catenin signal pathway in hepatocellular carcinoma cells.PKF-118-310,a pathway inhibitor,was added to further verify the role of Wnt/β-catenin pathway in the inhibitory effect of curcumin on invasion and metastasis of human liver cancer cells.CRISPR/Cas9 technique was adopted to knock out Bclaf1 gene to further clarify the regulatory role of Bclaf1 in the mechanism of curcumin inhibiting invasion and metastasis of human liver cancer cells.The transplanted tumor model of human hepatocellular carcinoma cell line HepG2 in nude mice was established and HepG2 cells were injected into tail vein to simulate lung metastasis of tumor cells,which were divided into negative control group,positive control group(5FU)and curcumin experimental group(20,40,60 μM),through measure the tumor volume to detect the inhibitory effect of curcumin on transplanted tumor in nude mice,and to observe the effect of curcumin on lung metastasis by HE staining of lung tissue and counting the number of pulmonary nodules.The expression of β-catenin,p-β-catenin,TCF4 and c-Myc was detected by Western blot technique,and the expression of Bclaf1,MMP2 and MMP9 was detected by immunohistochemical method.Results: The CCK8 results showed that the proliferation of human hepatoma cells HepG2 and SK-Hep-1 was inhibited by curcumin in a time-and dose-dependent manner.The scratch healing experiment showed that the migration ability of human hepatoma cells decreased with the increase of curcumin concentration.Transwell assay showed that the number of invading cells across the chambers decreased with increasing of curcumin concentration.Western blot assay showed that the expression of MMP2 and MMP9,which are invasion and metastasis-associated proteins,was down-regulated in different curcumin concentration groups,and curcumin could inhibit the expression of Wnt/β-catenin signaling pathway related proteins,and the inhibitory effect of curcumin on the expression of β-catenin,p-β-catenin,TCF4 and c-Myc was further enhanced by the addition of pathway inhibitor PKF-118-310,meanwhile,curcumin inhibited the expression of Bclaf1 in human hepatocellular carcinoma cells.Compared with the control group,the migration ability and invasive ability of hepatoma cells treated with curcumin after stable knockout of Bclaf1 were further decreased,and the expression of Wnt/β-catenin-related proteins was significantly down-regulated,and the expression of MMP2 and MMP9 was further decreased.Curcumin inhibited the growth of transplanted tumor in nude mice and significantly inhibited the lung metastasis of tumor cells with reduced the expression of Wnt/β-catenin pathway-related proteins and down-regulated expression of Bclaf1,MMP2 and MMP9 in tumor tissue.Conclusion: Curcumin can inhibit the invasion and metastasis of human hepatocellular carcinoma cells.The mechanism may inhibit Bclaf1 expression,thereby regulating Wnt/β-catenin pathway.