摘要
目的探讨抗增殖蛋白2(PHB2)在非小细胞肺癌细胞系A549对厄洛替尼(Erl)敏感性中的作用及其机制。方法在A549细胞中转染PHB2小干扰RNA(siPHB2),观察抑制PHB2表达对Erl诱导的细胞增殖和凋亡的影响。利用MitoTracker染色和感染绿色荧光蛋白-微管相关蛋白(GFP-LC3)观察微管相关蛋白(LC3)和线粒体共定位,EdU实验检测细胞增殖,平板集落检测细胞集落形成能力,TUNEL实验检测细胞凋亡,Western blot检测PHB2和LC3Ⅱ蛋白表达水平,用相应试剂盒检测线粒体膜电位、细胞色素c含量和呼吸链复合物Ⅰ/Ⅱ/Ⅴ活性。结果与siPHB2组和siCtrl+Erl组相比,siPHB2+Erl组EdU阳性的细胞数显著减少(P<0.05),集落形成数显著减少(P<0.05),TUNEL阳性的细胞数显著增加(P<0.05),线粒体膜电位显著降低(P<0.05),线粒体呼吸链复合物Ⅰ/Ⅱ/Ⅴ活性均显著降低(P<0.05),线粒体内细胞色素c减少(P<0.05),而细胞质内细胞色素c的增加(P<0.05)。结论抑制PHB2改善A549细胞对Erl的敏感性,其机制可能与抑制PHB2介导的线粒体自噬有关。
Objective To explore the effects of prohibitin 2(PHB2)on sensitivity of non-small cell lung cancer cell line A549 to erlotinib(Erl)and its potential mechanisms.Methods RACK1-specific small interfering RNA was transfected in A549 cells for knocking-down of PHB2.The effects of PHB2 inhibition on cell proliferation and apoptosis induced by Erl were observed.The colocalization of microtuble-associated protein light chain 3 alpha(LC3)and mitochondria was visualized by MitoTracker staining and green fluorescent protein-microtuble-associated protein light chain 3 alpha(GFP-LC3)transfection.Cell proliferation was detected by 5-ethynyl-2′-deoxyuridine(EdU)staining.Cell colony formation was evaluated by colony forming assay.Apoptotic index of A549 cells was evaluated by TUNEL.Western blot was used to measure the expressions of PHB2 and LC3Ⅱ.Mitochondrial transmembrane potential,cytochrome c and respiratory chain complexⅠ/Ⅱ/Ⅴactivity were analyzed by the commercially available kits.Results Compared with the siPHB2 and siCtrl+Erl group,the EdU-positive A549 cells and the number of cell colonies decreased significantly(P<0.05),while the TUNEL-positive A549 cells increased significantly(P<0.05)in the siPHB2+Erl group.In addition,compared with the siPHB2 and siCtrl+Erl group,mitochondrial transmembrane potential and respiratory chain complexⅠ/Ⅱ/Ⅴactivity decreased significantly(all P<0.05)and the levels of cytochrome c increased in mitochondrial fractions(P<0.05)and decreased in cytosolic fractions(P<0.05)in the siPHB2+Erl group.Conclusions PHB2 inhibition significantly improves sensitivity of A549 cells to Erl,which may be explained by inhibition of PHB2-mediated mitochondrial autophagy.
作者
张婧
杨自更
蔡文琴
曹维维
韦红梅
薛茜茜
吴宾
ZHANG Jing;YANG Zigeng;CAI Wenqin;CAO Weiwei;WEI Hongmei;XUE Xixi;WU Bin(Department of Nutrition,General Hospital of Xinjiang Military Command,Urumqi 830000;Department of Nuclear Medicine,General Hospital of Xinjiang Military Command,Urumqi 830000;Health Examine Center,General Hospital of Xinjiang Military Command,Urumqi 830000;The Fourth Department of Recuperation,Dalian Rehabilitation and Recuperation Center,Dalian 116000,China)
出处
《基础医学与临床》
2024年第3期325-332,共8页
Basic and Clinical Medicine
基金
新疆维吾尔自治区自然科学基金(2022D01C644,2023D01C94)
新疆军区总医院喀喇昆仑基金(2022JC002)。
关键词
抗增殖蛋白2
厄洛替尼
非小细胞肺癌
增殖
凋亡
prohibitin 2
erlotinib
non-small cell lung cancer
proliferation
apoptosis