The Research Of The Mechanism Underlying PGE₂Receptor EP2Activates Src/EGFR Signal Transduction Pathway Up-regulates Snail Protein Promoting The Invasiveness In Hepatocellular Carcinoma Cells

Background:Malignant tumor is one of the leading causes of death. The mortality of liver cancer is second in China. Since the absence of an effective chemoprevention or systematic treatment, the prognosis of HCC is very poor. Therefore, to explore the molecular mechanisms of HCC is extremely important.Prostaglandin E2(PGE2), a predominant metabolic product of cyclooxygenase-2(COX-2), exerts its biological functions via interaction with four kinds EP receptors on the cell surface membrane to promote the carcinoma cells growth, invasion and migration. PGE2has been shown to affect numerous tumorigenic progressions, such as hepatocellular carcinoma.Tumor invasion and metastasis are characterized by epithelial-mesenchymal transition (EMT). Snail, as one of key inducers of EMT, was found to play a major role in promoting tumor cell migration and invasion in many cancer types, such as ovarian cancer, colorectal cancer and so on.Our previous studies have shown that PGE2could significantly enhance HCC cell invasion and migration through upregulation of Snail expression level, however, the detailed mechanisms through which PGE2regulates Snail protein expression remains to be further defined. In this study, Huh-7cells have been treated with PGE2, EP2receptor agonist, EGFR inhibitor, PI3K inhibitor to detect the expression level of Snail, we aim to clarify the the mechanism underlying PGE2receptor EP2up-regulates Snail protein.Objectives:To investigate PGE2through EP2receptor up-regulates Snail protein expression and its related mechanism in hepatocellular carcinoma cells.Methods:1. The hepatocellular carcinoma Huh-7cells were cultured in conventional approach.2. Huh-7cells were treated with exogenous PGE2, cells migration and invasion was examined by Transwell.3. Huh-7cells were treated with exogenous PGE2and EP2receptor agonist Butaprost, Snail protein expressions were examined by Western blot.4. Huh-7cells were treated with Src inhibitor PP2, EGFR inhibitor AG1478, PI3K inhibitor LY294002, mTOR inhibitor PP242, Snail protein expressions were examined by Western blot.5. Huh-7cells were treated with Butaprost for0-60min, the alteration of Phospho-EGFR, Phospho-Akt, Phospho-mTOR were examined by Western blot.Results:1. Cell migration was found to increase by192%when the cells were treated with10μM PGE2for12h; Cell invasion was found to increase by186%when the cells were treated with10μM PGE2for24h.2. Huh-7cells were treated with10μM PGE2for24h, the level of Snail was increased by120.15%(P<0.01) compared with the control group. 3. Huh-7cells were treated with5μM Butaprost for24h, the level of Snail was increased by174.15%(P<0.01) compared with the control group.4. Butaprost-induced Snail expression was decreased about89.61%(P<0.01)by the10μM Src inhibitor PP2;67.92%(P<0.01) by the5μM EGFR inhibitor AG1478;56.63%(P<0.01) by the10μM PI3K inhibitor LY294002and99%(P<0.05) by the5μM mTOR inhibitor PP242compared with the butaprost treatment group.5. Butaprost-induced Phospho-Akt expression was decreased about85.69%and75.45%(P<0.05) by the10μM Src inhibitor PP2and5μM EGFR inhibitor AG1478, represtively, compared with the Butaprost treatment group.6. Huh-7cells were treated with5μM Butaprost for60min, the level of Phospho-EGFR、Phospho-Akt、Phospho-mTOR were increased by91.54%.134.67%、100.24%(P<0.05), represtively, compared with the control group.Conclusion:PGE2might up-regulate the expression level of Snail through EP2receptor in Huh-7cells, which was partly related to the Src/EGFR/Akt/mTOR signaling pathway.