Effects Of Histone Deacetylase Inhibitors Combined With FOXO1 Inhibitor On Hepatoma Cells And Its Molecular Mechanism

【objective】FOXO1 is indispensable in the process of epithelial-mesenchymal transition and autophagy induced by histone deacetylation inhibitors.It provides theoretical and experimental support for the combination of FOXO1 inhibitor and HDACIs in the prevention and treatment of liver cancer.【Methods】Part Ⅰ: Firstly,to explore whether HDACis can induce liver cancer EMT and autophagy.Then,the effects of HDACis on FOXO1,ULK1 expression was examined.We used hepatocellular carcinoma cell line Hep G2 and PLC as experimental subjects.After HDACIs(SAHA、Na B)induction treatment,we used gene expression profiling to detect differential expression of autophagy-related genes,and confirmed that HDACIs induced autophagy and EMT in liver cancer by increasing the level of FOXO1,ULK1.Then,the expression of EMT marker proteins(Vim,Snail,Fn)and the expression of autophagy-related proteins(LC3,P62)were analyzed by WB and RT-PCR.Cell migration and invasion ability was measured by cell scratch assay and Transwell chamber assay. Part II: Defining the key role of FOXO1 and ULK1 in HDACIs-induced autophagy and EMT.After antagonizing FOXO1 and ULK1 treatment,the effects of HDACIs(SAHA、Na B)induced hepatoma cell EMT and autophagy were analyzed by transfection technique,transmission electron microscopy and WB assay.Part III: To find out the effects of FOXO1 inhibitor combined with HDACIs(SAHA、Na B)on EMT? autophagy and apoptosis of hepatoma cells.Part IV: To explore the molecular mechanism of the combination of FOXO1 inhibitor and HDACIs(SAHA、Na B)to inhibit EMT in liver cancer.After treatment of liver cancer cells with FOXO1 inhibitor for a short time,the expression of p-Smad2/3 was detected by WB,the nuclear localization of Smad2/3 was detected by immunofluorescence,and the binding of Smad2/3 to Snail promoter was detected by EMSA.Part V: To explore the mechanism by which HDACIs regulates FOXO1.After treatment of liver cancer cells with SAHA and Na B in a short period of time,the expression of p-AMPK was detected by WB,and the nuclear localization of AMPK protein was detected by immunofluorescence.Finally,WB was used to detect the effects of AMPK inhibitor on EMT of liver cancer.【Results】1.HDACIs can induce EMT and autophagy in liver cancer cells and up-regulate the expression of FOXO1 and ULK1.2.HDACIs induces EMT and autophagy in hepatocellular carcinoma cells by up-regulating the expression of FOXO1 and ULK1.3.The mechanism by which FOXO1 inhibitors antagonize EMT in liver cancer mainly inhibits Smad2/3 phosphorylation,reduces Smad2/3 entry into the nucleus,and interferes with the binding of Smad2/3 to the Snail promoter.4.HDACIs regulate autophagy and EMT of liver cancer through the AMPK/FOXO1/ULK1 pathway.5.FOXO1 inhibitors combined with HDACIs can inhibit HDACIs-induced autophagy and EMT,and promote HDACis-induced apoptosis of hepatoma cells.【Conclusion】1.HDACIs regulates autophagy and EMT of liver cancer through the AMPK/FOXO1/ULK1 pathway.2.FOXO1 inhibitor combined with HDACIs can inhibit HDACIs-induced hepatoma autophagy and EMT,and promote HDACIs-induced hepatoma cell apoptosis.