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探讨氯化两面针碱(Nitidine chloride,NC)通过影响POLD1基因启动子甲基化率及调节转录调控因子p53和E2F的特异性结合对肝癌SMMC-7721细胞增殖和凋亡的影响。该研究采用CCK8,焦磷酸测序,RT-PCR,Western blot等技术方法论证了NC通过影响POLD1基因启动子甲基化率,间接调控POLD1基因转录活性,从而降低了DNA聚合酶δ的合成效率,抑制肝癌SMMC-7721细胞的增殖。不同剂量的NC(0.5,1.0,2.0 mg/m L)处理肝癌细胞SMMC-7721 48 h后,细胞增殖抑制率逐渐增大,分别为21.1%,35.2%,92.1%,呈明显的剂量依赖性。POLD1基因启动子的甲基化水平整体上升。经方差分析,与对照组相比,1.0,2.0 mg/m L实验组的E2F结合位点甲基化率随浓度增加而增高(F=10.21,P<0.05),差异有统计学意义,0.5 mg/m L实验组与对照组相比无明显差异(P=0.694)。而p53结合位点甲基化率随NC浓度增加呈下降趋势,实验组与对照组有明显差异(F=9.76,P<0.05),其中2.0 mg/m L实验组有显著差异(P<0.01)。实验组POLD1 mRNA和蛋白p125随NC浓度增加而下降,采用秩和检验,X2分别为40.19,36.65,P<0.05,1.0、2.0 mg/m L实验组与对照组相比,差异有统计学意义。结果显示氯化两面针碱可能通过调节SMMC-7721细胞POLD1基因的甲基化水平,影响其启动子转录调控因子的作用,从而抑制了POLD1基因表达,降低DNA聚合酶δ活性,抑制肝癌细胞增殖的作用。
To investigate the effects of nitidine chloride (NC) on the proliferation and apoptosis of hepatocellular carcinoma SMMC-7721 cells by affecting the promoter methylation of POLD1 gene and regulating the specific binding of transcriptional regulators p53 and E2F. In this study, CCK8, Pyrosequencing, RT-PCR and Western blot were used to demonstrate that NC indirectly controls the transcriptional activity of POLD1 by affecting the promoter methylation rate of POLD1 gene, thereby reducing the efficiency of DNA polymerase δ synthesis, Inhibit the proliferation of hepatoma SMMC-7721 cells. The proliferation inhibition rates of SMMC-7721 cells treated with different dosages of NC (0.5,1.0,2.0 mg / mL) for 48 h were 21.1%, 35.2% and 92.1%, respectively, in a dose-dependent manner . The methylation level of POLD1 gene promoter increased overall. By means of analysis of variance, compared with the control group, the methylation rate of E2F binding site in 1.0, 2.0 mg / m L experimental group increased with increasing concentration (F = 10.21, P <0.05), with a significant difference of 0.5 mg / m L experimental group compared with the control group no significant difference (P = 0.694). The methylation rate of p53 binding sites decreased with the increase of NC concentration. There was a significant difference between the experimental group and the control group (F = 9.76, P <0.05), and the 2.0 mg / m L experimental group was significantly different (P <0.01 ). The experimental group POLD1 mRNA and protein p125 decreased with the NC concentration increased by rank sum test, X2 were 40.19,36.65, P <0.05,1.0,2.0 mg / m L experimental group compared with the control group, the difference was statistically significant . The results showed that nitidine chloride may inhibit the expression of POLD1 gene, decrease the activity of DNA polymerase δ, and inhibit the proliferation of hepatocellular carcinoma cells by regulating the methylation level of POLD1 gene in SMMC-7721 cells and affecting its promoter transcriptional regulatory factor Role.