Rab26负性调控Nrf2增强肺癌耐药细胞对奥希替尼的敏感性

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

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1μM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1μM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。

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 1μM 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 1μM 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.