沉默UVRAG对白血病细胞K562线粒体自噬的影响

Effect of Silencing UVRAG on Mitophagy in Leukemia Cells K562

目的:探讨紫外线抵抗相关基因(UVRAG)对白血病细胞K562线粒体自噬的影响。方法:用不同浓度线粒体自噬诱导剂羰基氰化物间氯苯腙(CCCP)诱导K562细胞6、12和24 h,利用CCK-8法检测细胞活力;将K562细胞分为NC、UVRAG-siRNA、UVRAG-siRNA+CCCP和CCCP共4组,采用Western blot检测UVRAG蛋白质表达情况;流式细胞学技术检测活性氧、线粒体结构完整性的变化情况;Western blot检测自噬相关蛋白P62、LC3-Ⅱ/LC3-Ⅰ的表达情况。结果:与NC组相比,UVRAG-siRNA转染组UVRAG蛋白表达显著下降(P<0.01);与CCCP组相比,U VRAG-siRNA+CCCP组活性氧减少(P<0.05),线粒体结构破坏减少(P<0.05),LC3-Ⅱ/LC3-Ⅰ蛋白表达下降(P<0.01),P62蛋白表达上升(P<0.05);与NC组相比,UVRAG-siRNA组P62蛋白、LC3-Ⅱ/LC3-Ⅰ蛋白的表达,活性氧及线粒体结构完整性差异不显著(P>0.05)。结论:CCCP作用下,沉默UVRAG能抑制K562细胞的线粒体自噬。

Objective:To explore the effect of UVRAG on mitophagy in leukemia cells K562.Methods:K562 cells were induced with different concentrations of mitophagy inducer carbonylcyanide-m-chlorophenylhydrazone(CCCP)for 6,12 and 24 hours,and the cell viability was detected by the CCK-8 assay.K562 cells were divided into NC,UVRAGsiRNA,UVRAG-siRNA+CCCP,and CCCP group,while Western blot was used to detect the expression of UVRAG protein.Flow cytometry was used to detect the changes in reactive oxygen species(ROS)and mitochondrial structural integrity.The expressions of autophagy related proteins P62 and LC3-Ⅱ/LC3-Ⅰwere detected by Western blot.Results:Compared with NC group,the expression of UVRAG protein in UVRAG-siRNA group significantly decreased(P<0.01).Compared with CCCP group,in UVRAG-siRNA+CCCP group ROS,mitochondrial structure damage,and the expression of LC3-Ⅱ/LC3-Ⅰdecreased significantly(P<0.05,P<0.05,P<0.01),while the expression of P62 protein increased(P<0.05).Compared with NC group,the differences in the expressions of P62 and LC3-Ⅱ/LC3-Ⅰprotein,ROS,and mitochondrial structural integrity in UVRAG-siRNA group were not obvious(P>0.05).Conclusion:Under the treatment of CCCP,silencing UVRAG can inhibit mitophagy in K562 cells.