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中华肺部疾病杂志(电子版)

中华肺部疾病杂志(电子版) ›› 2024, Vol. 17 ›› Issue (03) : 349 -355. doi: 10.3877/cma.j.issn.1674-6902.2024.03.002

论著

Rab26负性调控Nrf2增强肺癌耐药细胞对奥希替尼的敏感性
殷国青1, 曾莉2, 贺斌峰2, 孙芬芬1,†()   
  1. 1. 400042　重庆，陆军特色医学中心呼吸科
    2. 400037　重庆，陆军（第三）军医大学第二附属医院全科医学科
  • 收稿日期:2024-03-21 出版日期:2024-06-25
  • 通信作者: 孙芬芬
  • 基金资助:
    重庆市自然科学基金面上项目(cstc2020jcyj-msxmX0096)

Rab26 enhances the sensitivity of lung cancer cells resistant to Osimertinib by negative regulation of Nrf2

Guoqing Yin1, Li Zeng2, Binfeng He2, Fenfen Sun1,†()   

  1. 1. Department of Respiratory, Army Medical Center of PLA, Chongqing, 400042
    2. Department of General practice, Second Affiliated Hospital of Army Medical University, Chongqing, 400037, China
  • Received:2024-03-21 Published:2024-06-25
  • Corresponding author: Fenfen Sun
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殷国青, 曾莉, 贺斌峰, 孙芬芬. Rab26负性调控Nrf2增强肺癌耐药细胞对奥希替尼的敏感性[J]. 中华肺部疾病杂志(电子版), 2024, 17(03): 349-355.

Guoqing Yin, Li Zeng, Binfeng He, Fenfen Sun. Rab26 enhances the sensitivity of lung cancer cells resistant to Osimertinib by negative regulation of Nrf2[J]. Chinese Journal of Lung Diseases(Electronic Edition), 2024, 17(03): 349-355.

目的

分析Rab26在奥希替尼耐药细胞中的表达及调控肺癌耐药细胞对奥希替尼敏感性的作用和机制。

方法

采用浓度梯度递增法诱导H1975细胞为奥希替尼耐药细胞株(H1975OR)。根据Western blot和qPCR检测H1975、H1975OR中Rab26与核因子红细胞2相关因子2(nuclear factor erythroid 2-related factor 2, Nrf2) mRNA及蛋白表达水平。对Rab26进行过表达慢病毒感染H1975OR细胞(Rab26 OE组)后给予1 μM奥希替尼处理48 h，通过CCK-8和TUNEL判断细胞活力和凋亡情况，用流式细胞术检测细胞ROS水平。用Western blot检测H1975和H1975OR中Nrf2蛋白经10 mM MG132处理8 h后的表达水平。对Nrf2进行siRNA转染H1975OR细胞后给予1 μM奥希替尼处理48 h，通过CCK-8判断细胞活力，利用流式细胞术检测细胞ROS水平。

结果

H1975OR中Rab26 mRNA和蛋白表达低于H1975细胞(P<0.05)，H1975OR中Nrf2蛋白表达高于H1975细胞(P<0.05)，H1975OR组和H1975组之间的mRNA水平差异无统计学意义(P>0.05)。奥希替尼干预后Rab26 OE组的细胞活力低于Vector组，ROS水平和凋亡水平较Vector组升高(P<0.05)。Rab26 OE组中Nrf2蛋白表达水平较Vector组降低，两组间Nrf2 mRNA差异无统计学意义(P>0.05)。经MG132处理，Nrf2蛋白水平在Vector组和Rab26 OE组中升高(P>0.05)。敲低Nrf2，奥希替尼干预增加H1975OR细胞中ROS水平，抑制其细胞活力。

结论

Rab26可负性调控Nrf2蛋白表达，促进奥希替尼诱导的ROS的过度产生，促进细胞凋亡，增强肺癌耐药细胞对奥希替尼的敏感性。

关键词: 支气管肺癌, Rab26, 奥希替尼耐药, Nrf2, 氧化应激
Objective

To explore the expression of Rab26 in osimertinib-resistant cells, and the role and mechanism of regulating lung cancer resistant cells′sensitivity to osimertinib.

Methods

Using a concentration gradient increasing method, induce H1975 cells to become osimertinib-resistant cell line (H1975OR). Western blot and qPCR were utilized to detect the expression levels of Rab26 and Nrf2 mRNA and proteins in H1975 and H1975OR. After overexpressing Rab26 through lentiviral infection in H1975OR cells (Rab26 OE group), continue to treat them with 1 μM osimertinib for 48 hours. Assess cell viability and apoptosis through CCK-8 and TUNEL, and evaluate ROS levels through flow cytometry. Use Western blot to assess Nrf2 protein expression levels in H1975 and H1975OR after treatment with 10 mM MG132 for 8 hours. Transfect H1975OR cells with Nrf2 siRNA, followed by treatment with 1 μM osimertinib for 48 hours, and evaluate cell viability using CCK-8 and ROS levels through flow cytometry.

Results

Rab26 mRNA and protein expression in H1975OR were significantly lower than in H1975 cells (P<0.05), while Nrf2 protein expression in H1975OR was significantly higher than in H1975 cells (P<0.05), with no significant difference at the mRNA level between the two groups (P>0.05). After osimertinib intervention, the cell viability of the Rab26 OE group was significantly lower than the control group (Vector), with a significant increase in apoptosis rate and ROS levels compared to the Vector group (P<0.05). The expression level of Nrf2 protein in the Rab26 OE group was significantly lower than in the Vector group, but there was no significant difference in Nrf2 mRNA between the two groups (P>0.05). After MG132 treatment, the Nrf2 protein level significantly increased in both the Vector group and Rab26 OE group (P>0.05). Knocking down Nrf2 significantly increased ROS levels in H1975OR cells after osimertinib intervention, while inhibiting cell viability.

Conclusion

Rab26 may promote the oxidative stress response induced by osimertinib by negatively regulating Nrf2, enhancing sensitivity of lung cancer resistant cells to osimertinib.

Key words: Bronchogenic carcinoma, Rab26, Osimertinib resistance, Nrf2, Oxidative Stress
图1 Rab26在H1975OR表达下调。注：A：倒置显微镜下H1975细胞和H1975OR细胞形态(比例尺：100 μM)；B: CCK-8检测H1975和H1975OR不同浓度Osi处理48 h后细胞存活率；C：H1975和H1975OR的IC50；D：qPCR检测H1975和H1975OR中Rab26 mRNA表达水平；E：Western blot检测H1975OR中Rab26蛋白表达水平；F：对E图的半定量分析。*：P<0.05.
图2 过表达Rab26增强H1975OR对奥希替尼敏感性。注：A：Western blot检测Rab26蛋白表达水平；B：对A图的半定量分析；C：CCK-8检测Vector和Rab26 OE细胞经1 μM奥希替尼(Osi)48 h处理后细胞存活率；D：Tunel检测Vector和Rab26 OE细胞经1 μM奥希替尼(Osi)48 h处理后细胞凋亡情况。*：P<0.05, ns：P>0.05
图3 Rab26促进ROS产生，增强H1975OR对奥希替尼敏感性。注：A：流式细胞仪检测奥希替尼处理后细胞中ROS平均荧光强度(mean fluorescence intensity, MFI)；B：CCK-8检测NAC预处理后经奥希替尼处理48 h后H1975OR/Rab26-OE细胞的存活率。*: P<0.05
图4 Rab26负性调控Nrf2蛋白表达。注：A：qPCR检测H1975和H1975OR细胞中Nrf2 mRNA表达水平；B：western blot检测H1975和H1975OR细胞中Nrf2蛋白表达水平；C：对B图的半定量分析；D：qPCR检测Vector和Rab26 OE细胞中Nrf2 mRNA及蛋白水平；E：western blot检测Vector和Rab26 OE细胞中Nrf2蛋白表达；F：对E图的半定量分析；G：Western blot检测Nrf2蛋白表达。Vector和Rab26 OE组细胞经10 μM MG132处理8 h; H：对G图的半定量分析。*：P<0.05, ns：P>0.05
图5 敲低Nrf2增强H1975OR细胞对奥希替尼敏感性。注：A：qPCR检测Nrf2 mRNA的表达水平；B：western blot检测Nrf2蛋白表达水平。Nrf2 siRNA和NC转染H1975OR细胞24 h；C：对B图的半定量分析；D：流式细胞术检测细胞中ROS水平；E：CCK-8检测细胞存活率。Nrf2 siRNA和NC转染H1975OR细胞24 h后，1 μM奥希替尼处理细胞48 h，Nrf2 siRNA和NC转染H1975OR细胞24 h后，1 μM奥希替尼处理细胞48 h。F：CCK-8检测细胞存活率，Vector和Rab26 OE细胞转染Nrf2 siRNA后，1 μM奥希替尼处理细胞48 h。*：P<0.05，ns：P>0.05
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