三氯乙酸对L-02细胞DNA甲基化转移酶1蛋白及mRNA表达的影响

Effects of trichloroacetic acid on protein and mRNA expression of DNMT1 in L-02 cells

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 effects of trichloroacetic acid （TCA）on protein and mRNA expression of DNMT1 in L-02 cells. Methods L-02 cells were exposed to the TCA in the medium at the doses of 0 （control）,0.1,0.3 and 0.9 minol/L respectively and then cultured for 24, 48, 72 hours. At the mean time, DNA methylation transfer 5-azadeoxycytidine（5-aza-dC, 5 p^inol/L） group,TCA-re group （treated with normal medium for 24 h after exposure to 0.9 mmol/L TCA） and human HepG2 cells group were set as the control groups. The InRNA expression of DNMTI was measured by real-time PCR,the protein expression of DNMT1 was tested by Western blotting. Results Compared with the control group,the InRNA expression levels of DNMT1 in all TCA- treated groups,5-aza-dC group and TCA-re group were significantly lower （P〈0.05）,the mRNA expression levels of DNMT1 in HepG2 cells group were significantly higher（P〈0.05）. Compared with the isodose TCA groups of 24 h,exeept the InRNA expression levels of DNMT1 in 0.1,0.9 minol/L TCA groups of 48 h,the InRNA expression levels of DNMT1 in other TCA concentrations groups of 48, 72 h were significantly lower （P〈0.05）. With the increase of the TCA exposure dose and time ,the mRNA expression of DNMTI showed a decreasing trend. Compared with the control group, except the protein expression levels of DNMT1 in 0.1 mmol/L TCA groups of 24, 48 h and 0.3 mmol/L TCA group of 24 h, the protein expression levels of DNMT1 in other TCA concentrations groups and 5-aza-dC group were significantly lower（P〈0.05）. But the protein expression levels of DNMT1 in TCA-re group and HepG2 ceils group had no significant change. Compared with the isodose TCA groups of 24 h, the protein expression levels of DNMT1 in all TCA-treated groups of 48, 72 h were significantly lower （P〈0.05）. With the increase of the TCA exposure dose and time, the protein expression of DNMT1 showed a decreasing trend, except that the protein expression levels of DNMT1 in all TCA treated groups of 24 h showed the trend of first rising and then falling with the increase of the TCA dose. Conclusion TCA can maintain or promote the low DNA methylation status of cultured cells by inhibiting the expression of DNMT1.