The Identification Of The Different Subtype Of EP₃ And The Related Cell Signal Transduction Pathway In Human Hepatocellular Carcinoma

Background:Recently,scientific researches indicate that COX-2 and its product PGE₂ play an important role in the process of tumorgenesis and development.PGE₂ produces a marked effect through EP receptors on the cell membrane.EP receptors are G-protein coupled receptors,which have four subtypes named EP₁,EP₂,EP₃ and EP₄,expressing on the human hepatocellular carcinoma.EP₃ receptor has eight different splicesomes which mediates different signal transduction pathway.This experiment, based on expression of EP₃ splicesomes on the the human hepatocellular carcinoma Huh7 cells,rudimently investigates the signal transduction pathways in human hepatocellular carcinoma cell proliferation mediated by EP₃ receptor,which establishes significant foundation for deeply studying the mechanisms of the promotion of PGE₂ on hepatocellular carcinoma cells growth. Objectives:This study was designed to investigate the subtype of the EP₃ and the related cell signal transduction pathway in human hepatocelluar carcinoma and to explore the molecular mechanism.Methods:1.Cell culture:Human hepatocellular carcinoma Huh7 cells were cultured in vitro as routine.2.The expression and splicesome type of EP₃ was determined by RT-PCR.3.The cell growth rate of Huh7 treated with sulprostone was detected by viability and growth rate(WST-1)assay.4.The level of intracellular cAMP concentration was measured by immunoassay Kit5.Western Blot analysis was used to detect the phosphorylation level of Akt and Erk after the treatment of sulprostone.Results:1.RT-PCR showed that the spliceosome of EP₃ expressed in Huh7 was EP₃(5).2.Cell viability and growth rate(WST-1)assay showed 1.0μmol/L,5.0μmol/L,10.0μmol/L sulprostone promoted the growth of Huh7 cells,and cell growth rates were increased 34.47%,28.25%,and 46.2%,respectively.3.Sulprostone increased intracellular cAMP in Huh7 cells.Treated by 0.0μmol/L,1.0μmol/L,5.0μmol/L,10.0μmol/L sulprostone,the intracellular cAMP concentration were 98.78pmol/L,145.9 pmol/L, 226.3 pmol/L and 478.95 pmol/L,respectively.4.The remarkable increase of Akt phosphorylation and decrease of Erk phosphorylation could be observed by Western blot after the treatment of sulprostone for 30 min.Conclusion:Our results suggested that in Huh7 cells,the subtype of the EP₃--EP₃(5)mediated a great increasing of the intracellular concentration of cAMP,which is partly related to the mechanism of Akt and Erk signaling pathway.