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目的 :探讨在肝细胞肝癌Huh-7细胞中前列腺素E2EP3各受体亚型对肿瘤细胞生长与侵袭的作用。方法:对常规培养的人肝细胞肝癌Huh-7细胞,瞬时转染EP3受体的4个亚型EP3-4R、EP3-5R、EP3-6R、EP3-7R,应用RT-PCR实验检测转染细胞EP3受体各亚型的m RNA表达水平;应用Western blot实验检测相关蛋白水平。对转染后的Huh-7细胞,分别给予不同浓度的EP3受体激动剂sulprostone作用24 h,用WST-8细胞增殖测定试剂检测细胞的增殖情况;应用划痕实验检测细胞的侵袭性;应用Western blot实验检测细胞内ERK蛋白磷酸化水平的变化。结果:RT-PCR及Western blot实验结果显示Huh-7细胞EP3受体表达水平明显增高。WST-8实验显示,经10μmol/L sulprostone处理24 h后,过表达EP3-4R、EP3-5R和EP3-7R亚型的细胞增殖率分别达到126.7%、124.0%、123.8%。划痕实验结果显示,过表达细胞的侵袭性分别增加到135.8%、123.5%、128.7%。Western blot显示,EP3-4R、EP3-5R和EP3-7R转染细胞内ERK磷酸化水平分别上调了54.8%、33.4%、44.3%。而过表达EP3-6受体亚型细胞的增殖率、侵袭率和胞内ERK磷酸化水平改变不明显。结论 :Huh-7肝癌细胞中,EP3-4R、EP3-5R及EP3-7R这3种受体亚型对细胞的生长及侵袭有显著促进作用,而EP3-6R亚型则无明显的促进作用。EP3受体促进肝癌细胞生长和侵袭的作用与ERK激活的现象一致。
Objective: To investigate the effect of prostaglandin E2EP3 receptor subtypes on the growth and invasion of tumor cells in hepatocellular carcinoma Huh-7 cells. METHODS: Four subtypes of EP3-4R, EP3-5R, EP3-6R and EP3-7R were transiently transfected into Huh-7 human hepatocellular carcinoma cell line, which were transfected by RT-PCR. The expression of m RNA in each subtype of EP3 receptor was detected by Western blot. The transfected Huh-7 cells were treated with different concentrations of sulprostone, an EP3 receptor agonist, for 24 h. The proliferation of Huh-7 cells was assayed by WST-8 cell proliferation assay. The cell invasiveness was evaluated by scratch assay. Western blot was used to detect the changes of intracellular ERK phosphorylation. Results: The results of RT-PCR and Western blot showed that the expression of EP3 receptor in Huh-7 cells was significantly increased. The results of WST-8 assay showed that the cell proliferation rates of EP3-4R, EP3-5R and EP3-7R subtypes were 126.7%, 124.0% and 123.8% respectively after treated with 10μmol / L sulprostone for 24 h. Scratch test results showed that the invasiveness of overexpressed cells increased to 135.8%, 123.5%, 128.7%, respectively. Western blot showed that phosphorylation of ERK in EP3-4R, EP3-5R and EP3-7R transfected cells were increased by 54.8%, 33.4% and 44.3%, respectively. However, the proliferation rate, invasion rate and intracellular ERK phosphorylation of EP3-6 receptor over-expression cells were not changed significantly. CONCLUSION: The three receptor subtypes EP3-4R, EP3-5R and EP3-7R in Huh-7 hepatocellular carcinoma cells significantly promote the growth and invasion of cells, while the subtype EP3-6R has no obvious promotion . The role of EP3 receptor in promoting hepatocellular carcinoma cell growth and invasion is consistent with the phenomenon of ERK activation.