前列腺素E2通过EP4受体调控甲状腺乳头状癌细胞增殖与侵袭的机制研究

Mechanism study of prostaglandin E2 regulating proliferation and invasion of papillary thyroid carcinoma cells via the EP4 receptor

摘要:

目的 探索前列腺素E2（PGE2）在甲状腺乳头状癌发生、发展中的作用及其机制。方法 选取甲状腺滤泡细胞（Nthy-ori 3-1）及人甲状腺乳头状癌细胞株TPC-1细胞进行培养。采用酶联免疫吸附试验检测2种细胞上清液PGE2含量，Western blotting及实时荧光聚合酶链反应检测2种细胞前列腺素E2受体4（EP4）表达情况。用0、1、2、5 μmol/L PGE2培养TPC-1细胞，CCK-8法检测细胞存活率，Western blotting检测EP4蛋白表达。0、5 μmol/L PGE2培养TPC-1细胞，CCK-8实验和Transwell实验检测细胞增殖和侵袭能力。0 μmol/L PGE2、5 μmol/L PGE2、5 μmol/L PGE2+5 μmol/L EP4受体激动剂（receptor agonist 2）、5 μ mol/L PGE2+5 μmol/L EP4受体抑制剂（L-161982）培养TPC-1细胞，CCK-8实验和Transwell实验检测细胞增殖和侵袭能力。结果 TPC-1细胞上清液PGE2含量高于Nthy-ori 3-1细胞（P <0.05）。TPC-1细胞EP4受体mRNA表达高于Nthy-ori 3-1细胞（P <0.05）。TPC-1细胞EP4受体蛋白表达高于Nthy-ori 3-1细胞（P <0.05）。5 μmol/L PGE2组细胞存活率较0 μmol/L PGE2组升高（P <0.05）。0 μmol/L PGE2组、5 μmol/L PGE2组24、48、72、96 h的细胞相对增殖率比较，结果 ①不同时间点细胞相对增殖率比较，差异有统计学意义（P <0.05）；②两组细胞相对增殖率结果比较，差异有统计学意义（P <0.05）；③两组细胞相对增殖率变化趋势比较，差异有统计学意义（P <0.05）。5 μmol/L PGE2组24 h细胞穿膜数高于0 μmol/L PGE2组（P <0.05）。2 μmol/L PGE2组、5 μmol/L PGE2组EP4蛋白相对表达量较0 μmol/L PGE2组升高（P <0.05）。0 μmol/L PGE2组、5 μmol/L PGE2组、5 μmol/L PGE2+5 μmol/L EP4 receptor agonist 2组、5 μmol/L PGE2+5 μmol/L EP4 L-161982组24 h、48 h、72 h、96 h的细胞相对增殖率比较，结果 ①不同时间点细胞相对增殖率比较，差异有统计学意义（P <0.05）；②各组细胞相对增殖率比较，差异有统计学意义（P <0.05）；③各组细胞相对增殖率变化趋势比较，差异有统计学意义（P <0.05）。5 μmol/L PGE2+EP4 receptor agonist 2组细胞穿膜数较5 μmol/L PGE2组增加（P <0.05），5 μmol/L PGE2+EP4 L-161982组细胞穿膜数较5 μmol/L PGE2组减少（P <0.05）。结论 PGE2通过EP4受体促进TPC-1细胞增殖及侵袭。

Abstract:

Objective To investigate the role of prostaglandin E2 (PGE2) in the pathogenesis of papillary thyroid carcinoma and its underlying mechanisms.Methods Human thyroid follicular cells (Nthy-ori 3-1) and papillary thyroid carcinoma cells (TPC-1) were cultured. The concentration of PGE2 in the cell culture supernatant was measured by ELISA. The expression of prostaglandin E2 receptor 4 (EP4) in both cell lines was detected by Western Blotting and qRT-PCR. TPC-1 cells were treated with 0 μmol/L, 1 μmol/L, 2 μmol/L, or 5 μmol/L PGE2, cell viability was assessed using the CCK-8 assay, and EP4 protein expression was detected by Western Blotting. TPC-1 cells were treated with 0 μmol/L or 5 μmol/L PGE2, and cell proliferation and invasion capabilities were evaluated using the CCK-8 assay and Transwell assay, respectively. Furthermore, TPC-1 cells were treated with 0 μmol/L PGE2, 5 μmol/L PGE2, 5 μmol/L PGE2 combined with 5 μmol/L EP4 receptor agonist (receptor agonist 2), or 5 μmol/L PGE2 combined with 5 μmol/L EP4 receptor inhibitor (L-161982), and cell proliferation and invasion capabilities were subsequently measured using the CCK-8 assay and Transwell assay.Results The PGE2 content in the culture supernatant of TPC-1 cells was higher than that of Nthy-ori 3-1 cells (P < 0.05). The mRNA expression level of the EP4 receptor in TPC-1 cells was higher than that in Nthy-ori 3-1 cells (P < 0.05). The protein expression level of the EP4 receptor in TPC-1 cells was higher than that in Nthy-ori 3-1 cells (P < 0.05). The cell viability in the 5 μmol/L PGE2 group was higher than that in the 0 μmol/L PGE2 group (P < 0.05). A repeated-measures ANOVA was conducted to compare the relative proliferation rates of cells in the 0 μmol/L and 5 μmol/L PGE2 groups at 24, 48, 72, and 96 h, and the results showed that the differences in relative proliferation rates among different time points were statistically significant (P < 0.05) and that the differences in relative proliferation rates between the two groups were statistically significant (P < 0.05). The differences in the change trends of relative proliferation rates between the two groups were also statistically significant (P < 0.05). The number of cells passing through the membrane in the 5 μmol/L PGE2 group at 24 h was higher than that in the 0 μmol/L PGE2 group (P < 0.05). The relative expression levels of EP4 protein in the 2 μmol/L and 5 μmol/L PGE2 groups were higher than those in the 0 μmol/L PGE2 group (P < 0.05). A repeated-measures ANOVA was performed to compare the relative proliferation rates of cells in the 0 μmol/L PGE2 group, 5 μmol/L PGE2 group, 5 μmol/L PGE2 + 5 μmol/L EP4 receptor agonist 2 group, and 5 μmol/L PGE2 + 5 μmol/L EP4 L-161982 group at 24, 48, 72, and 96 h, and the results showed that the differences in relative proliferation rates among different time points were statistically significant (P < 0.05) and that the differences in relative proliferation rates among the groups were statistically significant (P < 0.05). The differences in the change trends of relative proliferation rates among the groups were also statistically significant (P < 0.05). The number of cells passing through the membrane in the 5 μmol/L PGE2 + EP4 receptor agonist 2 group was higher than that in the 5 μmol/L PGE2 group (P < 0.05), while the number of cells passing through the membrane in the 5 μmol/L PGE2 + EP4 L-161982 group was lower than that in the 5 μmol/L PGE2 group (P < 0.05).Conclusion PGE2 promotes proliferation and invasion of TPC-1 cells via the EP4 receptor.