miR-28-5p通过抑制FSP1介导的铁死亡逆转肺腺癌细胞顺铂耐药的机制  

miR-28-5p reverses cisplatin resistance in lung adenocarcinoma cells by inhibiting FSP1-mediated ferroptosis

作　　者：王晓丹[1] 宋国庆[2] 姜丹 Wang Xiaodan;Song Guoqing;Jiang Dan(Dept of Second Thoracic Surgery,Shengjing Hospital of China Medical University,Shenyang 110000;Dept of First and Second Breast Tumor Surgery,Shengjing Hospital of China Medical University,Shenyang 110000)

机构地区：[1]中国医科大学附属盛京医院第二胸外科,沈阳110000 [2]中国医科大学附属盛京医院第一、第二乳腺肿瘤外科,沈阳110000

出　　处：《安徽医科大学学报》2023年第4期630-636,共7页Acta Universitatis Medicinalis Anhui

基　　金：辽宁省自然科学基金(编号:20180530020)。

摘　　要：目的 研究miR-28-5p对顺铂(DDP)耐药的A549肺腺癌细胞系(A549/DDP)的影响,并探讨其作用机制是否与铁死亡抑制蛋白1(FSP1)介导的铁死亡有关。方法 选择A549肺腺癌细胞和A549/DDP细胞作为研究对象。采用RT-qPCR法检测细胞中miR-28-5p的表达水平。通过CCK-8法和集落形成实验测定细胞增殖。miR-28-5p的靶基因通过荧光素酶报告基因和蛋白质印迹分析进行鉴定和验证。使用miR-28-5p模拟物或FSP1过表达质粒转染A549/DDP,评估细胞增殖情况,并采用透射电子显微镜评估线粒体形态,以及试剂盒测定细胞活性氧(ROS)、谷胱甘肽(GSH)和丙二醛(MDA)水平。建立了基于细胞系的异种移植模型,在体内评估miR-28-5p对肿瘤生长的影响。结果 与A549细胞相比,A549/DDP细胞中miR-28-5p的表达水平降低(P<0.001)。与A549细胞比较,A549/DDP细胞的细胞活力(F=49.542,P<0.001)和集落形成能力(t=4.412,P<0.01)增加。与miR-NC组相比,miR-28-5p组A549/DDP细胞的细胞活力(t=4.612,P<0.01)和集落数(t=4.503,P<0.01)均降低。在pGL3-FSP1 3′UTR-WT载体存在情况下,用miR-28-5p模拟物转染的细胞中荧光素酶活性降低(P<0.01)。此外,过表达miR-28-5p细胞中FSP1的表达水平被抑制。与载体+miR-28-5p组相比,FSP1+miR-28-5p组细胞活力、集落形成、细胞线粒体长度、GSH增加(P<0.01)且细胞凋亡、ROS产生、MDA形成降低(P<0.05)。体内实验显示,与miR-NC+DDP组相比,miR-28-5p+DDP组裸鼠体内形成的肿瘤大小和质量减少(P<0.05),并且Ki-67和FSP1蛋白表达降低(P<0.05)。结论 miR-28-5p逆转肺腺癌细胞顺铂的耐药机制可能与抑制FSP1介导的铁死亡有关。Objective To study the effect of miR-28-5p on cisplatin(DDP)-resistant A549 lung adenocarcinoma cell line(A549/DDP),and to explore whether its mechanism is related to ferroptosis suppressor protein 1(FSP1)-mediated ferroptosis.Methods The A549 lung adenocarcinoma cell line and A549/DDP cells were selected as the research objects.RT-qPCR method was used to detect the expression level of miR-28-5p in the cells.Cell proliferation was measured by CCK-8 method and colony formation assay.The target gene of miR-28-5p was identified and verified by luciferase reporter gene and Western blot analysis.A549/DDP was transfected with miR-28-5p simulant or FSP1 overexpression plasmid to evaluate cell proliferation,mitochondrial morphology was evaluated by transmission electron microscope,and the levels of reactive oxygen species(ROS),glutathione(GSH)and malondialdehyde(MDA)were measured by kit.A cell line-based xenograft model was established to evaluate the effect of miR-28-5p on tumor growth in vivo.Results Compared with A549 cells,the expression level of miR-28-5p in A549/DDP cells was significantly reduced(P<0.001).Compared with A549 cells,the cell viability(F=49.542,P<0.001)and colony forming ability(t=4.412,P<0.01)of A549/DDP cells increased significantly.Compared with miR-NC group,the cell viability(t=4.612,P<0.01)and colony number(t=4.503,P<0.01)of A549/DDP cells in miR-28-5p group significantly decreased.The luciferase activity decreased in the cells transfected with the miR-28-5p mimic,being significantly more so in the presence of the pGL3-FSP13′UTR-WT vector.In addition,the expression of FSP1 in cells overexpressing miR-28-5p was significantly suppressed.Compared with the Vector+miR-28-5p group,the FSP1+miR-28-5p group significantly increased cell viability and colony formation,cell mitochondrial length and GSH(P<0.01),and significantly increased cell apoptosis,ROS production and MDA formation decrease(P<0.05).In vivo experiments showed that compared with the miR-NC+DDP group,the size and weight of tumors formed i

关 键 词：miR-28-5p 肺癌 铁死亡抑制蛋白1 铁死亡 顺铂 

分 类 号：R734.2[医药卫生—肿瘤]