应用5-氮杂胞苷对甲状腺乳头状癌细胞株P14^(ARF)基因启动子区去甲基化干预的研究

Study of 5-Aza-Cd R induced demethylation of P14^(ARF) gene and its expression in papillary thyroid carcinoma cell line

目的观察5-氮杂胞苷对PTC细胞株P14ARF基因甲基化状态的影响。方法用不同浓度梯度的5-氮杂胞苷培养PTC细胞株;分别采用实时定量PCR、Western-bloting检测不同浓度5-氮杂胞苷处理甲状腺乳头状癌细胞株后P14ARFm RNA及其蛋白的表达情况;采用甲基化特异性PCR检测不同浓度5-氮杂胞苷处理PTC细胞株后P14ARF基因的甲基化情况;采用CCK8法检测5-氮杂胞苷处理后的PTC细胞株的增殖情况。结果实时定量PCR检测随着5-氮杂胞苷浓度增加及处理时间的延长,PTC细胞株P14ARFm RNA表达水平逐步上调(P<0.05);P14ARF蛋白的表达升高(P<0.05);甲基化特异性PCR检测经5-氮杂胞苷处理后的PTC细胞株中P14ARF基因的甲基化情况下降,出现去甲基化(P<0.05);采用CCK8法检测经5-氮杂胞苷处理后的PTC细胞株增殖速度减慢(P<0.05)。结论 P14ARF在PTC是存在甲基化的,5-氮杂胞苷可以逆转P14ARF的甲基化状态,抑制PTC细胞株的增殖,发挥抗肿瘤作用,为临床靶向治疗甲状腺乳头状癌提供分子生物学的依据。

Objective To observe effects of 5-Aza-Cd R on P14^ ARFgene methylation statue in papillary thyroid carcinoma cell line. Methods Different concentrations（ 0. 1 mol/L,1 mol/L,10 mol/L） of 5-Aza-Cd R were used to culture papillary thyroid carcinoma cell line; real-time quantitative PCR and Western-bloting were used to detect the expression of P14^ARF m RNA and protein in thyroid papillary carcinoma cell lines treated with different concentrations of 5-azacytidine; methylation-specific PCR was used to detect the methylation of P14^ ARF gene after 5-azacytidine treatment of PTC cell lines. The proliferation of PTC cell lines treated with 5-azacytidine was detected by CCK8 assay. Results Real-time PCR showed that P14 ^ARF m RNA was up-regulated and P14 ^ARF was up-regulated with the concentration increase of 5-azacytidine and prolongation of treatment time（ P〈0. 05）. The methylation status of P14 ^ARF gene in PTC cell lines treated with 5-azacytidine decreased and demethylation was detected by PCR（ P〈0. 05）. The 5-azacytidine-treated PTC cells detected by CCK8 plant proliferation slowed down-trend（ P〈0. 05）. Conclusion P14 ARFgene is methylated in papillary thyroid carcinoma,5-Aza-Cd R can reverse the methylation status of P14^ ARF,and inhibit the proliferation of PTC cell lines and exert anti-tumor effects,so as to provide molecular biological basis for the clinical targeted therapy of papillary thyroid carcinoma.