氯化两面针碱通过甲基化率调控p53和E2F介导肝癌POLD1基因表达的初步研究

Preliminary Study on the Effect of methylation Mediated the Pathway of p53 and E2F Acting on Liver Cancer POLD1 Gene Expression by Nitidine chloride

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 SMMC-7721 cell proliferation and apoptosis of Nitidine chloride （ nitidine chloride, NC） on POLD1 gene methylation and regulation of transcription factor p53 and E2F specifically binding to hepatoma SMMC-7721 cell. RTPCR, Pyrosequencing, and Western blot were used to test the POLD1 gene expression and to study the transcription activity of POLD1 by methylation on the treatment groups. After being starved for 48 h, SMMC-7721 cells treated with NC （0.5,1.0,2.0 mg/L）, NC significantly inhibiting the proliferation of SMMC-7721 cell lines was gradually increased,the rate of cell proliferation inhibition was 21.1% ,35.2% and 92.1% ,and it is a dose-dependent manner. Pyrosequencing found that POLD1 gene promoter methylation rates were markedly elevated. Analysis of ANOVO showed that methylation was obviously higher in the 1.0 mg/mL and 2.0 mg/mL experimental group than control group in the E2F binding sites of POLD1 promoter,F = 10.21 ,P 〈0.05. However,in the 0.5 mg/mL experimental group,there was no statistically significant difference,P = 0. 694. On the contrary, the methylation of p53 binding sites was lower than control group, F = 9.76, P 〈 0.05. Comparison in experimental groups, the difference was statistically significant in 2. 0 mg/mL experimental group,P 〈 0.01. RT-PCR result showed that POLD1 gene mRNA expressions and encoded protein p125 were both on the decline with the increase of concentration of NC. Wilcoxon was used,compared with control group,the 1.0 mg/mL and 2.0 mg/mL experimental groups have statistically significant difference,X2 were 40.19,40.19 P 〈0.05. But in the 0.5 mg/mL experimental＇ group, there was no statistically significant difference. Nitidine chloride participated in regulating POLD1 gene methylation levels of liver cancer,particularly enhanced the POLD1 gene promoter methylation rates and mediated the transcription activity of POLD1 gene, thus inhibiting the activity of DNA polymeraseδ and play the role in inhibiting hepatocarcinoma proliferation.