| 许永杰,陈万青.RNF8促进表皮生长因子受体野生型非小细胞肺癌吉非替尼耐药的机制分析[J].肿瘤学杂志,2023,29(12):1005-1010. |
| RNF8促进表皮生长因子受体野生型非小细胞肺癌吉非替尼耐药的机制分析 |
| Mechanism of RNF8 Facilitating Gefitinib Resistance in Non-Small Cell Lung Cancer with Wild-Type EGFR |
| 投稿时间：2023-08-14 |
| DOI：10.11735/j.issn.1671-170X.2023.12.B003 |
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| 中文关键词: 非小细胞肺癌 野生型表皮生长因子受体 吉非替尼 耐药 RNF8 Akt |
| 英文关键词:non-small cell lung cancer wild-type epidermal growth factor receptor Gefitinib resistance RNF8 Akt |
| 基金项目:北京协和医学院“中央高校基本科研业务费”项目（3332022031） |
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| 中文摘要: |
| 摘 要：［目的］ 探讨RNF8在携带野生型表皮生长因子受体（epidermal growth factor receptor，EGFR）非小细胞肺癌吉非替尼（Gefitinib）耐药中的作用及机制。［方法］ 通过RNAi法敲低RNF8，通过CRISPR/Cas9方法敲除RNF8，采用细胞敏感性实验和细胞克隆形成实验法评估敲低或敲除RNF8的A549细胞对Gefitinib的敏感性变化；采用Western blot法检测细胞中p-EGFR、EGFR、p-Akt、Akt和RNF8的表达水平；以裸鼠异体成瘤模型评估在体内敲除RNF8对Gefitinib敏感性的影响；以药物浓度逐步递增法构建对Gefitinib耐药的A549稳定细胞系，通过药物敏感性实验检测敲低RNF8的耐药细胞系对Gefitinib敏感性变化。［结果］ 在A549细胞中敲低RNF8，与对照组相比，不同浓度（2、5、10 μmol/L）的Gefitinib对细胞的抑制率均显著增强（12.21%±1.68% vs 45.48%±1.35%；25.13%±1.57% vs 60.93%±1.40%；36.67%±5.89% vs 78.92%±2.36%）（P均<0.001）。在不同浓度（5、10 μmol/L）Gefitinib处理下，与对照组相比，RNF8敲除的A549细胞克隆形成数量显著减少（45.00±16.05 vs 26.00±4.18；34.33±9.82 vs 11.67±1.21）（P均<0.001）。机制上，在A549细胞中敲低或敲除RNF8，Gefitinib对细胞中Akt磷酸化水平的抑制作用增强；过表达RNF8，Gefitinib对细胞中Akt磷酸化水平的抑制作用减弱。在裸鼠异体移植模型中，Gefitinib处理后，与对照组相比，RNF8敲除组小鼠肿瘤体积显著缩小［(450.98±98.54) mm3 vs (163.48±37.72) mm3，P<0.001］。在Gefitinib抵抗的A549细胞中用不同的siRNA敲低RNF8，Gefitinib对细胞的增殖抑制率均显著增强（P均<0.001），且敲低RNF8重新诱导Gefitinib对细胞中Akt磷酸化的抑制。［结论］ RNF8通过调节Akt的磷酸化激活促进野生型EGFR非小细胞肺癌细胞Gefitinib耐药。 |
| 英文摘要: |
| Abstract：［Objective］ To investigate the effect of RNF8 on Gefitinib resistance in non-small cell lung cancer (NSCLC) with wild-type epidermal growth factor receptor (wtEGFR) and the underlying mechanism. ［Methods］ RNF8 was knocked down by RNAi and knocked out by the CRISPR/Cas9 in human lung cancer A549 cells. Cell sensitivity assay and colony formation assay were performed to evaluate the changes in sensitivity to Gefitinib upon RNF8 silence or knockout in A549 cells. Western blot was conducted to detect the expression levels of p-EGFR， EGFR， p-Akt， Akt， and RNF8 in A549 cells. A tumor xenograft mouse model was established in Balb/c-null nude mice to assess the effect of RNF8 knockout on sensitivity to Gefitinib in vivo. Gefitinib-resistant A549 cell lines were established with stepwise dose-escalation continuous exposure. The effects of RNF8 down-regulation on the sensitivity of Gefitinib-resistant A549 cells were evaluated through cell sensitivity assays. ［Results］ Compared to the control group， knockdown of RNF8 significantly enhanced the inhibition rate of A549 cells treated with Gefitinib at different concentrations（2， 5， 10 μmol/L） (12.21%±1.68% vs 45.48%±1.35%； 25.13%±1.57% vs 60.93%±1.40%； 36.67%±5.89% vs 78.92%±2.36%， all P<0.001). Compared to the control， the number of colonies was significantly reduced in RNF8-knocked out A549 cells treated with different concentrations of Gefitinib (5， 10 μmol/L) (45.00±16.05 vs 26.00±4.18； 34.33±9.82 vs 11.67±1.21， all P<0.001). Knock down or knock out of RNF8 enhanced the inhibition of Akt phosphorylation by Gefitinib， while overexpression of RNF8 reduced the inhibitory effect of Gefitinib on Akt phosphorylation in A549 cells. For in vivo assay， RNF8KO xenografts were highly sensitive to Gefitinib ［(450.98±98.54) mm3 vs (163.48±37.72) mm3， P<0.001］. Knocking down RNF8 using two different siRNAs in Gefitinib-resistant A549 cells significantly enhanced Gefitinib’s inhibitory ability on cell proliferation (all P<0.001) and re-induced the inhibition of Akt phosphorylation by Gefitinib. ［Conclusion］ RNF8 promotes Gefitinib resistance in wtEGFR NSCLC by modulating Akt activity. |
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