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目的 Rab25在非小细胞肺癌(non-small cell lung cancer,NSCLC)等多种肿瘤中过度表达,提示其可能在NSCLC的发生发展及耐药形成中发挥重要作用。为进一步探讨Rab25的功能及其在NSCLC酪氨酸激酶抑制剂(epidermal growth factor receptor tyrosine kinase inhibitors,EGFR-TKIs)耐药形成中的作用,建立稳定慢病毒介导的shRNA靶向干扰Rab25基因人非小细胞肺癌厄洛替尼耐药细胞PC9/ER稳定株。方法实时定量PCR(real-time PCR,RT-PCR)检测PC9/ER及PC9细胞中Rab25基因mRNA的相对表达情况。筛选出Rab25基因的RNA干扰(RNA interference,RNAi)有效靶序列,合成靶序列的Oligo DNA并构建GV248-shRNA-Rab25慢病毒载体,酶切和测序鉴定正确后,经病毒包装,感染PC9/ER细胞,经嘌呤霉素筛选稳定表达细胞株。RT-PCR鉴定PC9/ER的表达,CCK-8检测对厄洛替尼的敏感性。结果 PC9/ER细胞中Rab25基因的mRNA表达水平显著高于PC9细胞。构建的重组慢病毒质粒经测序鉴定正确。RT-PCR证实,干扰Rab25后,PC9/ER细胞株中Rab25表达水平明显降低,抑制率为88.3%。通过传代10次后,PC9ER-Rab25i稳定细胞株中Rab25基因的mRNA表达水平显著低于阴性对照组,P<0.05。PC9ERRab25i稳定细胞株的IC_(50)为(2.133±0.222)μmol/L,显著低于阴性对照组的(6.375±0.799)μmol/L,P=0.007。结论成功构建了Rab25-shRNA慢病毒表达载体,建立了稳定抑制Rab25基因表达的人NSCLC厄洛替尼耐药细胞PC9/ER,初步验证Rab25基因能够改善肺癌EGFR-TKIs获得性耐药,为进一步研究Rab25在NSCLCEGFR-TKIs获得性耐药的机制及逆转其获得性耐药提供了可靠的细胞模型。
Objective Rab25 is overexpressed in many tumors such as non-small cell lung cancer (NSCLC), suggesting that Rab25 may play an important role in the development of NSCLC and the formation of drug resistance. To further investigate the function of Rab25 and its role in the resistance of NSCLC to EGFR-TKIs, we established a stable lentivirus-mediated shRNA targeting Rab25 gene Non-small cell lung cancer erlotinib-resistant cell PC9 / ER Strain. Methods The relative expression of Rab25 mRNA in PC9 / ER and PC9 cells was detected by real-time PCR (RT-PCR). The effective target sequence of RNA interference (RNAi) of Rab25 gene was screened and Oligo DNA of the target sequence was synthesized and the GV248-shRNA-Rab25 lentiviral vector was constructed. After identification and sequencing, the virus-infected PC9 / ER Cells were stably transfected with puromycin. The expression of PC9 / ER was detected by RT-PCR and the sensitivity of CCK-8 to erlotinib was detected. Results The mRNA expression of Rab25 in PC9 / ER cells was significantly higher than that in PC9 cells. The constructed recombinant lentiviral plasmid was identified by sequencing. The results of RT-PCR showed that the expression of Rab25 in PC9 / ER cell line was significantly decreased after interfering with Rab25, the inhibition rate was 88.3%. After passage 10 times, the expression level of Rab25 gene in PC9ER-Rab25i stable cell line was significantly lower than that in negative control group (P <0.05). The IC50 of PC9ERRab25i stable cell line was (2.133 ± 0.222) μmol / L, which was significantly lower than that of the negative control group (6.375 ± 0.799) μmol / L, P = 0.007. Conclusion Rab25-shRNA lentiviral vector was successfully constructed and human NSCLC erlotinib-resistant PC9 / ER cells stably inhibiting Rab25 gene expression were established. The preliminary validation of Rab25 gene could improve acquired resistance of lung cancer EGFR-TKIs. To study the mechanism of acquired resistance of Rab25 in NSCLCEGFR-TKIs and to provide a reliable cell model to reverse its acquired resistance.