Loss Of E-cadherin Upregulates The Phosphorylation Level Of GSK-3β In Human Hepatoma Cells

BackgroundHuman heptoma (HCC) is one of the most devastating malignancies in the China,also has a low five-year survival rate, and seriously threats human’s health and life.Tumor metastasis is process more than one step or one level. The premise ofmalignant tumor metastasize includes tumor cell adhesion, migration and attackability, and cell adhesion is more important.E-cadherin is the member of the cadherin family. Participating in the celladhesion and adjusting the cell proliferation-related signals are its main functions, andit is related to cell invasion and metastasis. Loss of E-cadherin can enhance the abilityof invasion and migration in breast cancer cells. Glycogen Synthase Kinase3β(GSK-3β) is a protein kinase, and it can inhibit glycogen synthase. It is the keycomponent of PI3K/Akt and Wnt/beta-catenin signaling, and has played an importantrole in the signaling pathway.Our evidences demonstrated that loss of E-cadherin downregulates expression ofGSK-3β in HepG2cells. But the regulation between E-cadherin and GSK-3β is notclear at present. In this study, HepG2cells were infected by lentivirus to gain stablecell lines dealing with PI3K inhibitor LY294002. Western Blot and RT-PCR were used to observe the possible signal transduction pathways.ObjectiveTo explore the molecular regulatory mechanism of loss of E-cadherin inductingGSK-3β phosphorylation deactivated in HCC.Methods1. Cell culture: Human heptoma cells HepG2and human embryonic kidneyHEK-293cells were all cultured in conventional canditions as routine. The lentivirusinfected stable cells HepG2-empty and HepG2-E-cadherin-shRNA were all culturedin vitro with puromycin.2. HepG2cells were infected by lentivirus to gain stable cells HepG2-empty andHepG2-E-cadherin-shRNA, and the expression level of E-cadherin was examined byWestern Blot analysis.3. The activity of GSK-3β was examined by Western Blot analysis in cytoplasmof HepG2-empty and HepG2-E-cadherin-shRNA cells.4. The mRNA level of Egr-1was tested by RT-PCR in HepG2-empty andHepG2-E-cadherin-shRNA cells.5. The mRNA level of PTEN was tested by RT-PCR, and the expression ofPTEN was examined by Western Blot analysis in HepG2-empty andHepG2-E-cadherin-shRNA cells.6. The phosphorylation level of Akt was examined by Western Blot analysis inHepG2-empty and HepG2-E-cadherin-shRNA cells.7. HepG2-E-cadhein-shRNA cells were treated with PI3K inhibitor LY294002(10μM), the phosphorylation level of GSK-3β was examined by Western Blotanalysis. Results1. Contrasted the blank, the expression level of E-cadherin downregulated82.54%(P<0.01) in HepG2-E-cadhein-shRNA cells.2. The phosphorylation level of GSK-3β upregulated298.93%(P<0.01) inHepG2-E-cadhein-shRNA cells compared with HepG2-empty cells.3. Contrasted the blank, the mRNA level of Egr-1dropped to66.89%(P<0.01)in HepG2-E-cadhein-shRNA cells.4. The mRNA level of PTEN dropped to76.14%(P<0.01), and the expressionlevel of PTEN downregulated to53.77%(P<0.01) in HepG2-E-cadhein-shRNA cellscompared with the control group.5. Contrasted the blank, the phosphorylation level of Akt upregulated218.98%(P<0.01) in HepG2-E-cadhein-shRNA cells.6. With10μM LY294002(PI3K inhibitor) treatmented for4h,8h,12h,16h and24h, the phosphorylation level of GSK-3β rised up to47.74%(P<0.01),58.16%(P<0.01),63.55%(P<0.01),60.38%(P<0.01) and62.18%(P<0.01) compared thegroup treated with DMSO.Conclusion1. E-cadherin downregulated with high phosphorylation level of GSK-3β inHepG2-E-cadherin-shRNA cells, revealed that the lentivirus have efficient RNAieffect.2. E-cadherin downregulated with low level of PTEN, low mRNA level of PTEN andEgr-1in HepG2-E-cadherin-shRNA cells, revealed that loss of E-cadherin inhibits thefunction of Egr-1and PTEN via PI3K/Akt signaling in human hepatoma cells.3. With the stimulation of LY294002(PI3K inhibitors), the phosphorylationlevel of GSK-3β rised up, and E-cadherin upregulated with high phosphorylation level of Akt in HepG2-E-cadherin-shRNA cells. These evidences demonstrated thatloss of E-cadherin downregulates expression of GSK-3β via PI3K/Akt signaling inHepG2cells.