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目的探讨三氯乙酸(TCA)染毒对L-02细胞DNA甲基化转移酶1(DNMT1)蛋白及mRNA表达的影响。方法取处于对数生长期的正常肝细胞(L-02细胞),分别加入含0(对照)、0.1、0.3、0.9 mmo/L TCA的培养液继续培养24、48、72 h,同时,设DNA甲基化酶抑制剂5-氮杂胞苷(5-aza-d C,5μmol/L)处理组,TCA-re组(0.9 mmol/L TCA处理后换正常培养基继续培养24 h)和人肝癌(HepG2)细胞组作为对照。检测细胞DNMT1蛋白和mRNA的表达水平。结果与对照组比较,各浓度TCA染毒组及5-aza-d C处理组、TCA-re组L-02细胞DNMT1 mRNA的表达水平均较低,而HepG2细胞组DNMT1 mRNA的表达水平较高,差异均有统计学意义(P<0.05)。与相同剂量TCA染毒24 h比较,各浓度TCA染毒48、72 h后L-02细胞DNMT1 mRNA的表达水平均较低,除0.1、0.9 mmol/L TCA染毒48 h外,差异均有统计学意义(P<0.05)。且随着TCA染毒浓度的升高和染毒时间的延长,L-02细胞DNMT1 mRNA的表达水平均呈下降趋势。与0.9 mmol/L TCA染毒组比较,TCA-re组L-02细胞DNMT1 mRNA的表达水平较高,差异有统计学意义(P<0.05)。与对照组比较,各浓度TCA染毒组及5-aza-d C处理组L-02细胞DNMT1蛋白的表达水平均较低,除0.1 mmol/L TCA染毒24、48 h及0.3mmol/L TCA染毒24 h外,差异均有统计学意义(P<0.05);而TCA-re组L-02细胞和HepG2细胞组DNMT1蛋白的表达水平均无明显变化。与相同剂量TCA染毒24 h比较,各浓度TCA染毒48、72 h后L-02细胞DNMT1蛋白的表达水平均较低,差异均有统计学意义(P<0.05)。除TCA染毒24 h时L-02细胞DNMT1蛋白的表达水平随染毒浓度的升高而呈先升高后下降的趋势外,随着TCA染毒浓度的升高和染毒时间的延长,L-02细胞DNMT1蛋白的表达水平均呈下降趋势。与0.9 mmol/L TCA染毒组比较,TCA-re组L-02细胞DNMT1蛋白表达水平较高,差异有统计学意义(P<0.05)。结论 TCA体外染毒可能通过抑制DNMT1的表达维持或者促进细胞的DNA低甲基化状态。
Objective To investigate the effect of trichloroacetic acid (TCA) on DNA methyltransferase 1 (DNMT1) mRNA and protein expression in L-02 cells. Methods The normal hepatocytes (L-02 cells) in logarithmic growth phase were cultured in culture medium containing 0 (control), 0.1, 0.3, 0.9 mmo / L TCA for 24, 48 and 72 h, respectively. The DNA methylase inhibitor 5-azacytidine (5μmol / L), TCA-re group (0.9 mmol / L TCA for normal medium for 24 h) and Human hepatocellular carcinoma (HepG2) cell group served as a control. The expression level of DNMT1 protein and mRNA were detected. Results Compared with the control group, the expression of DNMT1 mRNA in L-02 cells of TCA-re treated group and 5-aza-d C treated group was lower than that of TCA-re treated group, while DNMT1 mRNA expression was higher in HepG2 cells , The differences were statistically significant (P <0.05). Compared with the same dose of TCA for 24 h, DNMT1 mRNA expression in L-02 cells was lower at 48 and 72 h after TCA exposure, except for 0.1 and 0.9 mmol / L TCA exposure for 48 h Statistical significance (P <0.05). With the increase of TCA concentration and exposure time, the expression level of DNMT1 mRNA in L-02 cells showed a decreasing trend. Compared with 0.9 mmol / L TCA group, DNMT1 mRNA expression in L-02 cells in TCA-re group was significantly higher than that in TCA-re group (P <0.05). Compared with the control group, the DNMT1 protein expression levels in L-02 cells in TCA-treated and 5-aza-d-treated groups were all lower than those in control group except for 24 h, 48 h and 0.3 mmol / L TCA exposure TCA exposure for 24 h, the differences were statistically significant (P <0.05); while TCA-re group DNMT1 protein expression levels in L-02 cells and HepG2 cells had no significant change. Compared with the same dose of TCA for 24 hours, DNMT1 protein expression in L-02 cells was lower at each concentration of TCA for 48 and 72 h (P <0.05). Except TCA, the expression level of DNMT1 protein in L-02 cells increased firstly and then decreased with the increase of the concentration of TCA. With the increase of TCA concentration and the prolongation of exposure time, L-02 cells DNMT1 protein expression levels showed a downward trend. Compared with 0.9 mmol / L TCA group, DNMT1 protein expression in L-02 cells of TCA-re group was significantly higher than that of 0.9 mmol / L TCA group (P <0.05). Conclusion TCA exposure in vitro may inhibit the expression of DNMT1 to maintain or promote DNA hypomethylation of cells.