The Function And Mechanism Of EP3 Receptor Subtypes In Promoting Hepatoma Huh-7 Cell Growth And Invasion

Backgroud:Worldwide, the incidence of cancer is facing grave problems for its elevating morbidity and mortality, of which, about half of the new liver cancer cases occurred in China, which leads to serious danger to the mankind. Clinically, lacking of effective early-diagnosis and therapy bring poor prognosis to liver cancer.Prostaglandin E2(PGE2) is a kind of inflammatory mediator, which has significant promoting effects on the proliferation and invasiveness of cancer cells. In hepatocellular carcinoma cell lines, PGE2 could bind with four kinds of E prostanoid receptors on the cell membrane, which are EP1 R, EP2 R, EP3 R and EP4 R, and influence the biological properties of hepatocellular carcinoma cell lines from different aspects.According to our previous results, we found that on the cell membrane of hepatocellular carcinoma Huh-7 cells exist four EP3 receptor subtypes: EP3-4, EP3-5, EP3-6 and EP3-7, but the function and mechanism of EP3 receptor subtypes in promoting hepatocellular carcinoma proliferation and invasiveness is still unclear.On the basis of our previous research work, we further analyzed the function and mechanism of EP3 receptor subtypes in promoting cell growth and invasion of Huh-7 cells. Objectives:To investigate the function and mechanism of EP3 receptor subtypes in promoting cell growth and invasion of hepatoma Huh-7 cells. Methods:1. Human hepatocellular carcinoma cells Huh-7 were cultured in a conventional method.2. Huh-7 cells were transiently transfected with EP3-4, EP3-5, EP3-6 and EP3-7 receptors.3. The expression of EP3 receptor in the transfected cells was detected by RT-PCR and western blot.4. The cells were treated with various concentrations of selective EP3 agonist sulprostone. The cell viability was detected in the transfected cells by WST-8.5. The scratch test was used to detect the invasion ability in transfected cells.6. Western blot was employed to detect the level of P-Erk. Results:1. The expression of EP3 receptor subtypes was markedly increased in transfected cells.2. Under the treatment of 10 μmol/L sulprostone for 24 hours, the cell growth rate of Huh-7 cells transiently transfected with EP3-4, EP3-5 and EP3-7 receptor was respectively increased to 126.7%, 124.0% and 123.8%.3. Under the treatment of 10 μmol/L sulprostone for 24 hours, the growth of invasion rate in cells over expressed of EP3-4, EP3-5 and EP3-7 receptor separately came to 135.8%, 123.5% and 128.7%.4. The concentration of intracellular P-Erk in Huh-7 cells over-expressed of EP3-4, EP3-5 and EP3-7 receptor increased by 54.8%, 33.4% and 44.3%.5. Over-expression of EP3-6 receptor cells had no significant difference in cell growth, cell invasion and the level of intracellular Erk phosphorylation. Conclusion:Our results demonstrate that EP3-4, EP3-5 and EP3-7 these three EP3 receptor subtypes mediate a great progress of the ability of proliferation and invasion in hepatocellular carcinoma cells Huh-7, whereas EP3-6 receptor does not. And these findings suggest that a negative feedback of balancing regulation mechanism may exist among EP receptors. Additionally, the function of EP3 receptor in accelerating cell growth and invasion of Huh-7 cells is in line with the activation of Erk.