组蛋白去乙酰化酶抑制剂对胶质瘤细胞增殖及MKK7表达的影响

目的 探讨组蛋白去乙酰化酶抑制剂（histone deacetylase inhibitor, HDACI）对胶质瘤细胞增殖及MKK7表达的影响。方法 体外培养神经胶质瘤细胞株U251，用脂质体转染MKK7-siRNA1、MKK7-siRNA2和对照siRNA，48 h后Western blot检测MKK7表达及JNK/c-Jun活性，BrdU掺入实验检测细胞增殖情况；用0.5 μM组蛋白去乙酰化酶抑制剂曲古菌素A（trichostatin A, TSA）处理细胞8 h，DMSO作对照，Western blot检测MKK7、p-JNK、JNK、p-c-Jun、c-Jun表达或磷酸化水平；用TSA分别处理细胞8、12、24 h，检测各时间点细胞增殖率；用其他组蛋白去乙酰化酶抑制剂包括伏立诺他（suberoylanilide hydroxamic acid, SAHA），丙戊酸（valproic acid, VPA），M344处理细胞检测MKK7表达及细胞增殖。结果 同对照组相比，沉默MKK7表达抑制了JNK/c-Jun活性和细胞增殖（P=0.008）；TSA处理细胞8 h显著抑制MKK7表达及JNK/c-Jun活性，SAHA、M344、VPA均显著抑制MKK7表达。TSA处理细胞8 h没有抑制细胞增殖，处理12 h显著抑制增殖（P=0.006）， 24 h抑制效应更明显（P=0.002）； SAHA（P=0.001）, M344（P=0.008）或VPA（P=0.002）处理细胞12 h均抑制了细胞增殖。结论 组蛋白去乙酰化酶抑制剂可通过抑制MKK7表达抑制神经胶质瘤细胞增殖。

Effects for Histone Deacetylase Inhibitors on Proliferation and MKK7 Expression in Glioma Cells

Objective To investigate the effects of histone deacetylase inhibitors (HDACIs) on the proliferation and MKK7 expression in glioma cells. Methods Glioma cell line U251 cultured in vitro was transfected with MKK7-siRNA1, MKK7-siRNA2 and control siRNA for 48h, respectively. Western blot was performed to test MKK7 expression and JNK/c-Jun activities. BrdU incorporation assays were performed to determine the cellular proliferation rate. Cells were treated with 0.5μM HDAC inhibitor Trichostatin A (TSA) for 8h, dimethyl sulfoxide (DMSO) as a control. Western blot was performed to test the expression or phosphorylation levels of MKK7, p-JNK, JNK, p-c-Jun and c-Jun. MTT assays were performed to determine cellular proliferation rate at 8, 12, 24h after TSA treatment. Other HDACIs, including suberoylanilide hydroxamic acid (SAHA), valproic acid (VPA) and M344, were used to test if they could inhibit MKK7 expression and cellular proliferation as well. Results Compared with the control group, the knockdown of MKK7 suppressed JNK/c-Jun activities and proliferation rate(P=0.008); HDACI (0.5 μM TSA) treatment for 8h remarkably reduced MKK7 expression and JNK/c-Jun activities, and all other HDACIs (SAHA, M344, VPA) could suppress MKK7 expression as well. TSA treatment for 8 h did not decrease the proliferation rate, while TSA treatment for 12h significantly inhibited proliferation rate (P=0.006), and treatment for 24h exhibited more inhibitive effects (P=0.002); SAHA (P=0.001), M344 (P=0.008) or VPA (P=0.002) inhibited cellular proliferation as well. Conclusion HDACIs could inhibit MKK7 expression and MKK7-mdiated proliferation in glioma cells.