摘要
目的 探讨RNA聚合酶2延长因子相关因子2（EAF2）对前列腺癌细胞增殖和迁移能力的抑制作用与细胞内细胞外信号调节激酶（ERK）磷酸化的关系。方法 采用RNA干扰（RNAi）方法干扰前列腺癌细胞C4-2和22Rv1中EAF2的表达，采用Western blot方法检测细胞内ERK磷酸化水平的变化；采用质粒转染的方法在293T细胞中过表达EAF2后检测ERK磷酸化水平的变化；分别采用溴脱氧尿嘧啶核苷（BrdU）标记法和Transwell小室法检测干扰EAF2表达对前列腺癌细胞增殖和迁移能力的影响，利用ERK抑制剂PD184352同时抑制ERK磷酸化后，进一步检测细胞增殖和迁移能力的变化。结果 干扰EAF2表达后ERK的磷酸化水平明显升高。过表达EAF2的表达后ERK的磷酸化水平进行性下降，在C4-2细胞中干扰EAF2的表达，检测到增殖细胞的百分比从（19.7±1.5）%升高至（30.8±0.8）%（P＝0.001）。但同时抑制细胞中ERK的磷酸化后，增殖细胞的百分比从（30.8±0.8）%降至（21.9±0.5）% （P＝0.013）。在C4-2细胞中干扰目标基因EAF2的表达后，检测到迁移细胞的数量从（104±12）个升高至（203±17）个（P＝0.002）。但同时抑制细胞中ERK的磷酸化后，迁移细胞的数量从（203±17）个降至（123±11）个（P＝0.011）。结论 EAF2的表达和ERK的磷酸化水平相关，EAF2可能通过调节ERK的磷酸化水平调控前列腺癌细胞增殖和迁移的能力。
Objective To investigate whether ELL-associated factor 2 （EAF2） inhibits prostate cancercell proliferation and migration by regulating level of extracellular regulated protein （ERK） phosphorylation.Methods After inhibiting expression of EAF2 by interfering RNA （RNAi） in C4-2 and 22Rv1 cells, the expression level of phosphorylation of ERK was assessed by Western blotting. After transfecting myc-EAF2 into 293T cells according to the manufacturer’s instruction, the expression level of phosphorylation of E RK was assessed by Western blotting. After knockdown of EAF2 by RNAi in C4-2 cells, proliferation and migration abilities were tested by either Bromodeoxyuridine incorporation assay （BrdU） or Transwell migration assay. To detect whether EAF2 affects cells proliferation and migration by targeting ERK phosphorylation, ERK inhibitor （PD184352） was used to inhibit ERK phosphorylation, then the BrdU and Transwell assays were repeated.Results The expression of phosphorylation-ERK （p-ERK） was significantly increased after transfection of small interfering RNA （siRNA） against EAF2, not total-ERK （t-ERK）. The expression of p-ERK was significantly decreased after transfection with myc-EAF2 plasmid. Moreover, proliferation was further decreased from （30.8±0.8）% to （21.9±0.5）% （P=0.001） after combined transfection of siRNA targeting EAF2 and ERK inhibitor （PD184352） compared to knockdown of EAF2 cells （P=0.013）. The migration was decreased from （203±17） cells to （123±11） cells （P=0.002） after combined transfection of siRNA targeting EAF2 and inhibitor of ERK （PD184352） compared to single knockdown of cells （P=0.011）.Conclusion These findings suggest that EAF2 regulates p-ERK level in prostate cancer cells and that EAF2 regulates cells’ proliferation and migration abilities via ERK phosphorylation.
出处
《中华实验外科杂志》
CSCD
北大核心
2017年第4期635-637,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金（81241027）
