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目的探讨Rab25在非小细胞肺癌(non-small cell lung cancer,NSCLC)厄洛替尼获得性耐药中的作用。方法传代培养PC9及其厄洛替尼耐药PC9/ER细胞,采用CCK-8法检测PC9/ER细胞的耐药指数。应用实时荧光定量PCR(quantitative real-time,q PCR)及Western blot检测对比同等厄洛替尼浓度(0.1、0.2、0.3μmol/L)处理条件下,Rab25在两种细胞中的mRNA及蛋白表达差异,并以免疫荧光法定位Rab25在细胞中的分布。用小干扰RNA(siRab25)转染PC9/ER细胞,并以流式细胞仪及CCK-8法分别检测Rab25敲减后PC9/ER细胞的增殖指数(proliferation index,PI)及厄洛替尼IC50值的变化。结果 PC9/ER细胞的耐药指数是68.82±21.42。相同厄洛替尼浓度处理48 h后,PC9/ER细胞Rab25 mRNA及蛋白水平均高于PC9细胞(P<0.05),增高的Rab25蛋白主要表达于细胞膜。而且PC9/ER细胞Rab25蛋白水平随厄洛替尼处理浓度(0.1、0.2、0.3μmol/L)递增而增高。siRab25在PC9/ER细胞中成功转染后,转染组的厄洛替尼IC50值及PI均明显低于对照组(P<0.05)。结论Rab25在NSCLC厄洛替尼获得性耐药细胞中高表达,起着促进耐药的作用。
Objective To investigate the role of Rab25 in acquiring acquired resistance to erlotinib in non-small cell lung cancer (NSCLC). Methods PC9 and ER9 / ER cells were subcultured and the drug resistance index of PC9 / ER cells was detected by CCK-8 assay. Using real-time quantitative PCR (qPCR) and Western blotting, the mRNA and protein expression of Rab25 in two kinds of cells were compared under the same concentration of erlotinib (0.1,0.2,0.3μmol / L) Difference, and immunofluorescence localization of Rab25 in the distribution of cells. PC9 / ER cells were transfected with small interfering RNA (siRNA) (siRab25). The proliferation index (PI) of PC9 / ER cells after Rab25 knockdown and flow cytometry and CCK-8 assay were used to detect the IC50 of erlotinib Value changes. Results The resistance index of PC9 / ER cells was 68.82 ± 21.42. At the same concentration of erlotinib for 48 h, the Rab25 mRNA and protein levels in PC9 / ER cells were higher than those in PC9 cells (P <0.05). The increased Rab25 protein was mainly expressed in the cell membrane. Moreover, the Rab25 protein level in PC9 / ER cells increased with the increasing concentrations of erlotinib (0.1,0.2,0.3μmol / L). After successful transfection of siRab25 in PC9 / ER cells, the IC50 and PI of erlotinib in transfection group were significantly lower than those in control group (P <0.05). Conclusion Rab25 is highly expressed in erlotinib-resistant NSCLC cells and plays a role in promoting drug resistance.