Eicosapentaenoic Acid(EPA) Inhibits Human Hepatoma Cells Invasion And Metastasis By Regulating The Epithelial-mesenchymal Transition

BackgroundHepatocellular carcinoma(HCC) is a kind of highly malignant solid tumors, and its invasion and metastasis are important biological deterioration characteristics of hepatoma for the most primary reason to death on the patients. Tumor cells invasion and metastasis consist complex multiple factors and mechanisms involved in the co-regulation process and it has been confirmed that Epithelial-mesenchymal transition(EMT) is one of the important molecular mechanisms of invasion and metastasis of hepatocellular carcinoma cells. As the key mechanism of the development of hepatoma, telomerase activity is directly regulated by human telomerase reverse transcriptase(h TERT).As the key molecule to maintain telomerase structure and function, h TERT is closely related to the development of EMT in tumor cells found in recent research. Eicosapentaenoic acid(EPA) is a member of the n-3 polyunsaturated fatty acids and previous studies have found that it can restrain the process of a variety of malignant tumors, but now its anti-cancer effect mechanism is not clear, especially the EPA on hepatoma cells invasion and metastasis ability has not yet been reported.Objectives1.Clear the impact of EPA to TGF-β1 induced SMMC-7721 cells proliferation and apoptosis of hepatoma; 2.Whether the EPA can reverse TGF-β1 induced hepatoma SMMC-7721 cells EMT; 3.Whether the EPA can affect the invasion and metastasis of hepatoma SMMC-7721 cells the EMT mediated; 4.To explore the role of h TERT in EPA affecting the EMT process.Part 1 The Impact of EPA to Hepatoma SMMC-7721 Cells Proliferation and ApoptosisUse different concentrations of EPA(0、25、50、100、200、300、400 μM) and TGF-β1(10 ng/ml)to interfere commonly human hepatoma SMMC-7721 cells and detect the changes of the hepatoma SMMC-7721 cells proliferation and apoptosis.Methods1.Observe the hepatoma SMMC-7721 cells growth under inverted microscope; 2.Detect the hepatoma SMMC-7721 cells proliferation rate by MTT; 3.Detect the hepatoma SMMC-7721 cells apoptosis by flow cytometry; 4.Detect the Bax、Bcl-2 protein expression of hepatoma SMMC-7721 cells by Western.Results1.Preliminary observation under a microscope shows that the EPA to hepatoma SMMC-7721 cells growth performs an inhibition effect; 2.Determined by MTT test results confirm that the proliferation rate after the EPA intervention to hepatoma SMMC-7721 cells gradually decreases and presents the concentration dependence of time; 3.Flow cytometry results show that after the EPA intervention to hepotoma SMMC-7721 cells on 48 h it performs apoptosis and the apoptosis rate and EPA concentration are positively correlated; 4.Western results show that the EPA intervention to hepotoma SMMC-7721 cells on 48 h performs up-regulated expression of pro-apoptosis protein Bax and down-regulated expression of anti-apoptosis protein Bcl-2.ConclusionThe EPA can inhibit hepatoma SMMC-7721 cells proliferation, promote its apoptosis and its inhibition effect has the concentration dependence of time.Part 2 The Impact of the EPA to TGF-β1 Induced Hepatoma SMMC-7721 Cells Invasion and Metastasis Ability10 ng/ml TGF-β1 stimulates hepatoma SMMC-7721 cells to induce EMT. Use 50 μM, 200 μM EPA to interfere TGF-β1 induced hepatoma SMMC-7721 cells for testing the changes of invasion and metastasis ability.Methods1.Observe the cells morphological changes of hepatoma SMMC-7721 cell under inverted microscope; 2.Detect the hepatoma SMMC-7721 cell E-cadherin and N-cadherin protein expression by Western; 3.Detect the mobility of hepatoma SMMC-7721 cells by scratch test; 4.Detect the invasion rate of hepatoma SMMC-7721 cells by Transwell; 5.Detect the MMP-2、MMP-9 expression of hepatoma SMMC-7721 cells by ELISA.Results1.Preliminary observation under a microscope performs that EPA can inhibit TGF-β1 induced hepatoma SMMC-7721 cells epithelial mesenchymal transition; 2.Western results prove that the EPA can reverse the TGF-β1 induced hepatoma SMMC-7721 cells epithelial mesenchymal transition; 3.Scratch experiment confirms that the EPA can significantly reduce the TGF-β1 induced hepatoma SMMC-7721 cells migration ability and the inhibition effect is concentration dependence of time; 4.Transwell experiment establishes that the EPA can significantly reduce the TGF-β1 induced hepatoma SMMC-7721 cells invasion ability and the inhibition effect is concentration dependence of time too; 5.ELISA test results show that the EPA can inhibit TGF-β1 induced hepatoma SMMC-7721 cells to secrete matrix-metalloproteinases MMP-2 and MMP-9.Conclusion10 ng/ml TGF-β1 can induce epithelial mesenchymal transition of the hepatoma SMMC-7721 cells. The EPA can reverse epithelial mesenchymal transition mediated by TGF-β1 and inhibit hepatoma SMMC-7721 cells on the release of MMP-2 and MMP-9 to reduce SMMC-7721 cells ability of invasion and metastasis.Part 3 The Impact of EPA to TGF-β1 Induced Hepotoma SMMC-7721 Cells Telomerase ActivityUse 50 μM and 200 μM EPA to act on TGF-β1 induced hepatoma SMMC-7721 cells to detect hepatoma SMMC-7721 cells telomerase activity and the expression of telomerase regulatory protein h TERT.Methods1.Detect hepatoma SMMC-7721 cells telomerase activity by ELISA; 2.Detect the expression of h TERT protein in hepatoma SMMC-7721 cells by Western; 3.Detect the expression h TERT m RNA in hepatoma SMMC-7721 cells by Real-time PCR; 4.Observe the fluorescence intensity of hepatoma SMMC-7721 cells h TERT by cell immunofluorescent assay.Results1.The EPA can inhibit TGF-β1 induced hepatoma SMMC-7721 cells telomerase activity, and the inhibition effect is concentration dependence of time; 2.The EPA can significantly reduce the TGF-β1 induced hepatoma SMMC-7721 cells h TERT protein expression; 3.The EPA can significantly reduce the TGF-β1 induced hepatoma SMMC-7721 cells h TERT m RNA expression; 4.Cell immunofluorescence results show that the EPA can reduce the TGF-β1 induced hepatoma SMMC-7721 cells h TERT fluorescence intensity.ConclusionThe EPA can inhibit TGF-β1 induced hepatoma SMMC-7721 cells h TERT protein and m RNA expression, and thus reduce SMMC-7721 cells ability of invasion and metastasis.h TERT may be an important target that the EPA develop its anti-cancer effect and inhibit hepatoma cells invasion and metastasis.