目的：探讨直接抑制细胞外信号调节激酶（extra cellular signal-regulated kinase, ERK）表达后,对肿瘤坏死因子相关的凋亡诱导配体(TNF related apoptosis inducing ligand, TRAIL)对黑素瘤细胞杀伤作用的影响及其作用机制。方法：用携带ERK特异或对照shRNA的慢病毒感染A375黑素瘤细胞，48 h后加入终浓度为100 μg/ml基因重组TRAIL蛋白继续培养6 h，收获细胞，碘化丙啶染色，用流式细胞术检测细胞周期、细胞凋亡和TRAIL受体的表达；Western blotting检测相关蛋白的表达水平。结果：亚型特异的ERK shRNA分别沉默ERK1或ERK2，导致A375细胞出现G1期阻滞（对照shRNA组的G1期细胞比例为71%，ERK1、ERK2、ERK1+2 shRNA组分别增加到85%、90%和81%，P<0.01）；S期细胞从对照组的11%减少到2%左右（P<0.001）、G2/M期细胞也从对照组的17%减少到3%（P<0.01）。细胞周期改变伴有P21、P27蛋白上调和Cyclin D1蛋白降低，但未见明显的细胞凋亡。经对照shRNA和TRAIL处理的细胞，仅有15%的凋亡率，而ERK shRNA和TRAIL联合处理则使凋亡率达到40%~60%（P<0.01）。抑制ERK蛋白表达能显著上调细胞表面TRAIL受体DR4的表达（从对照组32%增加到75%~80%，P<0.01），但DR5变化不明显。ERK抑制还明显降低A375细胞的葡萄糖转运受体1(Glut 1)和己糖激酶Ⅱ(HK-Ⅱ)的蛋白表达水平。结论：直接抑制ERK不仅可以抑制肿瘤细胞周期的进展，而且可以增强TRAIL对A375黑素瘤细胞的杀伤作用，其机制与上调细胞表面DR4的表达和干扰肿瘤细胞糖代谢有关。

Objective:To investigate the effect of extra cellular signal-regulated kinase （ERK） knockdown on melanoma cell apoptosis mediated by TNF related apoptosis inducing ligand (TRAIL) and the underlying mechanisms. Methods: A375 cells were infected with lentivirus carrying either scramble shRNA (SC shRNA) or ERK (shRNA) for 48 hours and then treated with recombinant TRAIL protein (100 μg/ml) for another 6 hours. Cells were harvested and stained with PI; Flow cytometry was used to detect cell cycle, apoptosis, and the expression of TRAIL receptor; Western blotting was used to measure the expression level of relevant proteins. Results: Knockdown of either ERK1 or ERK2 by isotype specific shRNA resulted in G1-phase growth arrest of A375 cells（cell percentageat G1-phase in control group was 71%, compared to 85%, 90%, 81% respectively in ERK1, ERK2 and ERK1+2 shRNA groups, P<0.01). Accordingly, the A375 cell percentage at S-phase was 11% in control group, compared to around 2% in various ERK shRNA groups (P<0.001); the G2/M cell percentage was 17% in control group, compared to the around 3% in various ERK shRNA groups (P<0.01). The change of cell cycle was accompanied by up-regulation of P21 and P27 proteins, and down-regulation of cyclin D1 level, however no obvious cell apoptosis was observed. Treatment of scramble shRNA together with TRAIL caused about 15% of cell apoptosis,in contrast, the combined treatment of ERK shRNA and TRAIL increased cell apoptosis rate up to 40%~60% (P<0.01). Silencing of ERK enhanced DR4 expression on TRAIL receptor (from 32% to 75%~80%, P<0.01), but not DR5 expression. Furthermore, directly silencing ERK resulted in inhibited expression of Glut 1 and hexokinase Ⅱ, which are involved in the unique glucose metabolism of tumor cells. Conclusion: Directly silencing ERK with specific shRNA inhibited tumor cell growth and enhanced tumor cell apoptosis mediated by TRAIL. A combination of increased DR4 expression and impaired glucose metabolism is likely to contribute to the synergistic effect seen with TRAIL and ERK shRNA treatment in melanoma cells.

国家自然科学基金资助项目(No.81241148)