Studies On E-cadherin Expression Inducement Of Prostaglandin E₂ In Hepg2 Cells And Its Molecular Mechanism

Background:Human hepatocellular carcinoma (HCC) is one of the most devastating malignancies in the world and its morbidity and mortality are still increasing year by year. About 300,000 people died of this disease every year in China, which ranks second only to lung cancer in the sequence of cancer mortality. The high mortality rate of HCC is caused by its recurrence and metastasis, which is one of the biggest obstacles in the treatment. The study of its mechanism has always been a hot topic at home and abroad.COX-2/PGE2 show high expression in many tumor tissues. COX-2 derived prostaglandin E2 (PGE2) can promote tumor growth by binding its receptors and activating signaling pathways which control cell proliferation, migration, apoptosis, and angiogenesis. Receently, Prostaglandin E2 (PGE2) is also found to play a major role in promoting tumor cell migration,invasion and metastasis.E-cadherin is the major component of intercellular connections, which mediates cell-cell adhesion and maintains cell structure and the stable morphology. Reduced expression of E-cadherin can possiblely repress cell-cell adhesion, make cell separation, result in cell amotio and diffusion, promote the local invasion of cancer cells, and then the distant metastasis occurs. Mariam Dohadwala has reported that PGE2, in autocrine or paracrine fashion, modulates transcriptional repressors of E-cadherin and thereby regulates COX-2-dependent E-cadherin expression in non–small cell lung cancer (NSCLC). Thus, blocking PGE2 production or activity may contribute to both prevention and treatment of NSCLC. This study was designed to investigate the effect of Prostaglandin E2 on the expression of E- cadherin in HepG2 cells and the related cell signal transduction pathway.Objective:To investigate the effect of Prostaglandin E2 on the expression of E- cadherin in HepG2 cells and the related cell signal transduction pathway and to explore the molecular mechanism.Methods:1. Cell culture: human hepatoma cells (HepG2) were cultured in vitro as routine.2. HepG2 cells were transfected with PCDNA3-COX-2, Western blot analysis was employed to detect E-cadherin expression.3. After HepG2 cells were treated with different concentration of PGE2 for 24h, the expression level of E-cadherin was detected by Western blot analysis.4. After HepG2 cells were treated with 20μM PGE2 for 24h,48h,72h, the E-cadherin mRNA and protein level were examined by reverse transcription-PCR( RT-PCR) and Western blot analysis respectively.5. Western Blot analysis was used to detect the phosphorylation level of p-Akt after HepG2 cells were treated with 20μM PGE2 for 0min,5min,15min,30min,45min,60min.6. PI-3K inhibitor LY294002 were added to culture media, the expression of E-cadherin was tested by Western blot analysis to determine whether Akt signaling pathway was involved in the mechanism exerted by PGE2.Results:1. After HepG2 cells were transfected with PCDNA3-COX-2, the expression level of E-cadherin was decreased by 30.5% when compared with PCDNA3 control transfection cells(P<0.05).2. After HepG2 cells were treated with extrinsic source 5,10,20μM PGE2, the expression level of E-cadherin was decreased by 22.31%,36.3% and 47.83% respectively when compared with control group(P<0.01).3. The level of E-cadherin mRNA was decreased by 21.78%,48.11% and 45.02% respectively and the protein level was decreased by 39.23%,55.56% and 77.76% respectively compared with control group after HepG2 cells were treated with 20μM PGE2 for 24h,48h and 72h.4. After HepG2 cells were treated with 20μM PGE2 for 0min,5min,15min,30min,45min,60min, Western Blot analysis showed that the phosphorylation level of p-Akt was increased 135.28%,192.68%,152.29%,152.96% and 129.67% respectively(p<0.01), and reached the highest level at 15 minutes.5. After the treatment with PI-3K inhibitor LY294002 in HepG2 cells, Western Blot analysis showed that the expression level of E-cadherin which was mediated by PGE2 returned to the basal level, and had significant difference when compared with PGE2 group; But the expression level of E-cadherin of LY294002 solitudely treatment group was still at the basal level.Conclusions:1. COX-2 overexpression transfected HepG2 cells show greatly decreased thelevel of E-cadherin expression. 2. PGE₂ can significantly inhibit the expression level of E-cadherin protein and mRNA in HepG2 cells in a dose and time dependent manner.3. LY294002 can partially block the effect of PGE₂ on E-cadherin expression. PGE2 can repress E-cadherin expression in HepG2 cells, which is probably related to the Akt signal transduction pathway.