PYK2介导H460肺肿瘤细胞失巢凋亡抗性的机制

摘要

[目的]研究蛋白激酶PYK2对悬浮状态下的肺癌细胞H460失巢凋亡抗性的作用和PYK2的下游通路蛋白。[方法]RNA干扰实验采用逆转录病毒载体介导的稳定表达系统降低PYK2蛋白的表达；细胞悬浮培养实验用polyHEMA培养方法：用PARP断裂带和DAPI荧光染色检测细胞凋亡情况；细胞侵袭能力用侵袭小室实验；磷酸化蛋白激酶水平用免疫印记实验检测。[结果]用RNA干扰沉默能够降低H460细胞中PYK2蛋白的表达。抑制PYK2的表达能够降低悬浮培养H460细胞增殖能力和促进细胞凋亡增加。此外，沉默PYK2的表达能使肿瘤细胞侵袭能力下降，并降低磷酸化Paxillin和Src的水平，但不能降低磷酸化JNK、AKT和ERK的水平。[结论]PYK2信号通路在H460肺癌细胞失巢凋亡抗性中起重要作用,Paxillin和Src可能是PYK2的下游通路蛋白。%[Objective] To investigate roles of protein kinase PYK2 in anoikis resistance of lung carcinoma H460 cells, and the downstream signal transduction of PYK2. [Methods] The stable knockdown of PYK2 expression was achieved by RNA interference experiments mediated by Retrovirus vector. The experiment of suspension culture was used by polyHEMA culture system. PARP cleavage and DAPI dye were used to detect the cell apoptosis. Transwell chamber was used to determine the cell invasive ability. The phosphorylated level of protein kinase was investigated by Western blot. [Results] The level of PYK2 protein in H460 cells reduced by RNA interference system. The inhibition of PYK2 expression could decrease the proliferation of H460 tumor cells and increase the apoptosis of H460 tumor cells. Knockdown of PYK2 expression could reduce the cell invasive ability and reduce the phosphorylated level of Paxillin and Src protein kinase, but not the phosphorylated level of JNK,AKT,and ERK. [Conclusion] PYK2 plays an important role in anoikis resistance of H460 cell, and Paxillin and Src may be the downstream signals oi PYK2.