普鲁卡因对结肠癌细胞中孕酮及脂联素受体3甲基化的影响

Effects of procaine on the methylation of PAQR3 gene in colon cancer cells

目的研究孕酮及脂联素受体3(PAQR3)在结肠癌细胞中的表达水平及甲基化状态,检测普鲁卡因逆转PAQR3甲基化的作用。方法通过RT-PCR检测PAQR3在结肠癌细胞株(SW620、COLO205及SW480)中的表达水平;甲基化特异性聚合酶链反应检测PAQR3在结直肠癌细胞中的甲基化状态及药物处理后的甲基化状态;Western blotting检测不同浓度普鲁卡因处理SW480细胞中PAQR3蛋白的表达。结果PAQR3在SW620结肠癌细胞中的表达量高于COLO205和SW480(P<0.05)。3种结肠癌细胞株可见PAQR3基因甲基化特异性扩增产物条带,且甲基化百分比比较,差异有统计学意义(P<0.05)。SW620细胞较SW480和COLO205细胞中PAQR3甲基化百分比高(P<0.05);普鲁卡因处理后3种结肠癌细胞株可见甲基化特异性扩增产物条带和非甲基化特异性扩增条带,且3种细胞中PAQR3甲基化百分比降低(P<0.05)。对照组与实验组PAQR3蛋白相对表达量比较,差异有统计学意义(P<0.05)。与对照组比较,实验组PAQR3蛋白相对表达量升高(P<0.05)。结论PAQR3在结直肠癌中低表达,可能与其发生启动子甲基化有关。普鲁卡因可部分逆转结直肠癌细胞中PAQR3甲基化状态,上调PAQR3蛋白表达量。

Objective To investigate the expression of progestin and adipoq receptor family member 3(PAQR3)in colon cancer cell lines and detection of the aberrant methylation status in its promoter,and to detect the demethylating effect of procaine hydrochloride(PCA).Methods The mRNA expression of PAQR3 in colon cancer cell lines(SW620,COLO205,SW480)was detected by realtime-PCR(RT-PCR).The promoter methylation status of PAQR3 in colon cancer cells and the methylation status after drug treatment were detected by methylationspecific PCR.The expression of PAQR3 protein in procaine hydrochloride treating SW480 cells was detected by western blot.Results The expression of PAQR3 was decreased in three colon cancer cells,the difference was statistically significant(P<0.05),and the expression of SW620 was lower than COLO205 and SW480(P<0.05);the methylationspecific amplification product bands of PAQR3 gene were observed in all three colon cancer cell lines,and the difference in methylation percentage was statistically significant(P<0.05).Compared with SW620 cells,the percentage of PAQR3 methylation was significantly lower in SW480 and COLO205 cells(P<0.05);methylation-specific amplification product bands and non-methylation-specific amplification bands were observed in the three colon cancer cell lines treated with procaine hydrochloride,and the percentage of PAQR3 methylation was significantly decreased in the three cells,and the difference was statistically significant(P<0.05).There was a statistically significant difference in the relative expression of PAQR3 protein between the control group and the procaine hydrochloride treatment group(P<0.05).Compared with the control group,the PAQR3 protein in the procaine hydrochloride treatment group was increased(P<0.05).Conclusions The relative expression of PAQR3 mRNA in colon cancer cells was significantly decreased,which may be related to its promoter methylation.PCA can partially reverse the methylation status of PAQR3 in colon cancer cell lines and up-regulate the expression of PAQR3 protein.