Effects And Mechanism Of SGK3 On Liver Cancer Cell Epithelial-Mesenchymal Transition

Background:Hepatocellular carcinoma(HCC) is one of the most common liver cancer and the second most the frequent cause of cancer death. Metastasis is the primary reason for the death of patients. Therefore, it is very important to search the mechanism of HCC metastasis.The EMT is a process which changes the cell phenotype from epithelial to mesenchymal states. Studies indicate that EMT has a critical role in cancer metastatic progression. Therefore, study the liver cancer EMT provides a new road to explore liver cancer metastasis.Micro RNAs(mi RNAs) are short, ~22 nt noncoding RNAs which can have important regulatory roles in life processes. There is generally accepted that mi RNAs have an important role in cancer pathogenesis, and could either promotes or suppress tumour. There are many mi RNAs regulate HCC development and some mi RNAs are associated with EMT,including mi R-155. Our prior research demonstrated a clear role for mi R-155 in promoting epithelial-mesenchymal transition and cancer stem cell phenotypes.The phosphatidylinositol 3 kinase(PI3K) pathway is one of the most important pathways in cancer metabolism and growth. SGK is similar with AKT suggesting that SGKs may function similarly to AKT in cancer development. SGKs have been reported to be involved in the regulation of cell growth, proliferation, survival, and migration. But there is no evidence demonstrating a direct relationship between SGK3 and EMT. Therefore, SGK3 may act as a important targets for the liver cancer metastasis treatment of liver cancer metastasis.Part I The role of SGK3 in liver cancer EMTObjective:To determine whether up-regulate or down-regulation SGK3 have a role in HCC cells EMT.Methods:1.Use high-expressing lentiviral vector or SGK3-si RNA to up-regulate or down-regulation the expression of SGK3 in HCC cell culture lines SMMC-7721 and Huh-7.2. Use Western blot to determine the expression level of EMT markers E-cadherin, Vimentin and N-cadherin.3. Take Transwell migration assay and Matrigel invasion assay to determine the capacity of migration and invasion ability.4. Wound healing assays was used to determine the Wound healing ability.Results:Depletion of SGK3 led to the up-regulation of E-cadherin and the down-regulation of both N-cadherin and Vimentin in SMMC-7721 and Huh-7 cells. Overexpression of SGK3 in SMMC-7721 and Huh-7 cells led to the down-regulation of E-cadherin and the up-regulation of both N-cadherin and Vimentin. Silencing the expression of SGK3 decreased the invasion ability and migration ability in both SMMC-7721 and Huh-7 cells. Overexpression of SGK3 enhanced the invasion and migration ability of SMMC-7721 and Huh-7 cells. Overexpression of SGK3 increased the wound-healing capacity in both SMMC-7721 cells and Huh-7 cells capacity in both SMMC-7721 cells and Huh-7 cells.Conclusion:SGK3 has a important role in the EMT of HCC cells.Part II mi R-155 regulates EMT through targeted regulation of SGK3Objective:To determine whether up-regulate or down-regulation mi R-155 have a role in the expression of SGK3 and AKT1.Methods:1. Use mi R-155 mimic/mi R-155 inhibitor up-regulate or down-regulation the expression of mi R-155 in HCC cell culture lines SMMC-7721 and Huh-7.2. Use Western blot to determine the expression level of SGK3,p-AKT and AKT.Results:Down-regulation of mi R-155 decreased the expression of SGK3, but had no apparent effect on the level of p-AKT. Up-regulation of mi R-155 increased the expression of SGK3, but had no apparent effect on the level of p-AKT.Conclusion:Mi R-155 could regulate the expression of SGK3 and AKT1,but the effect on SGK3 is obvious than AKT1.Part III SGK3 regulates β-catenin signaling in HCC cellsObjective:To determine whether down-regulation SGK3 have a role in the expression ofβ-catenin.Methods:1. Use SGK3-si RNA to down-regulation the expression of SGK3 in HCC cell culture lines SMMC-7721 and Huh-7. Use Western blot to determine the expression level of Active β-catenin and β-catenin.2. Use proteasome inhibitor MG132 in the above cells. Use Western blot to determine the expression level of Active β-catenin and β-catenin.Results:Reduction of SGK3 expression using si RNA in SMMC-7721 and Huh-7 cells markedly reduced the level of total, as well as the active pool(dephosphorylated on Ser37 and Ser41), β-catenin. This effect of SGK3 depletion on β-catenin was blocked in the presence of the proteasome inhibitor MG132.Conclusion:SGK3 regulates β-catenin signaling in HCC cells...