HSPA5通过抑制铁死亡调控宫颈癌细胞的增殖和凋亡  

HSPA5 regulates proliferation and apoptosis of cervical cancer cells through inhibiting ferroptosis

作　　者：胡瑶蕊 杨柳[3] 裴尧 胡威[2] 杨春华[2] 张璐萍[1] HU Yaorui;YANG Liu;PEI Yao;HU Wei;YANG Chunhua;ZHANG Luping(Institute of Neurobiology,Binzhou Medical University,Shandong Yantai 264000,China;Shandong Technology Innovation Center of Molecular targeting and Intelligent Diagnosis and Treatment,Binzhou Medical University,Shandong Yantai 264000,China;Department of Paediatrics,Jinan Central Hospital,Shandong Jinan 250013,China)

机构地区：[1]滨州医学院神经生物学研究所,山东烟台264000 [2]滨州医学院山东省分子靶向智能诊疗技术创新中心,山东烟台264000 [3]济南市中心医院儿科,山东济南250013

出　　处：《现代肿瘤医学》2023年第11期1996-2002,共7页Journal of Modern Oncology

基　　金：山东省自然科学基金资助项目(编号:ZR2021MH141)。

摘　　要：目的:探讨热休克蛋白70蛋白5(heat shock 70 kDa protein 5,HSPA5)通过铁死亡对宫颈癌细胞增殖和凋亡的影响。方法:实时荧光定量PCR(qRT-PCR)和蛋白质印迹法(Western Blot,WB)检测HSPA5和谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)在宫颈癌和癌旁组织中的表达情况,通过TCGA数据库分析所有宫颈鳞状细胞癌和宫颈内膜腺癌(CESC)中HSPA5与患者总生存率间的相关性。体外培养人宫颈癌HeLa和SiHa细胞系,分别感染shHSPA5和shCtrl慢病毒,构建稳定的宫颈癌HSPA5敲低株,用qRT-PCR和WB检测HSPA5敲低之后铁死亡相关蛋白GPX4的表达情况。使用铁死亡抑制剂Ferrostatin-1(Fer-1)或激活剂Erastin处理细胞后,采用CCK-8和EdU检测细胞增殖情况;乳酸脱氢酶(LDH)试剂盒检测LDH含量;还原型谷胱甘肽(GSH)试剂盒和丙二醛(MDA)试剂盒分别测定GSH和MDA含量;流式细胞术检测细胞凋亡及活性氧(ROS)的情况。结果:与癌旁组织相比,宫颈癌组织中HSPA5与GPX4的表达显著升高,HSPA5高表达与患者预后不良有关;在HeLa和SiHa细胞中敲低HSPA5能够抑制细胞的增殖和促进细胞凋亡;同时细胞内ROS、MDA和LDH的水平上调;HSPA5敲低后GPX4的mRNA和蛋白表达水平同时降低,此外细胞内GSH的含量降低;铁死亡抑制剂Fer-1逆转了HSPA5敲低导致的ROS和LDH的含量升高并抑制细胞凋亡;铁死亡激活剂Erastin和HSPA5敲低联合增加了细胞凋亡并抑制细胞增殖。结论:HSPA5在宫颈癌组织中表达上调,通过敲低HSPA5可激活铁死亡从而抑制宫颈癌细胞的增殖和促进细胞凋亡。Objective:To investigate the effects of HSPA5 on the proliferation and apoptosis of cervical cancer cells through ferroptosis.Methods:Quantitative Real-time PCR(qRT-PCR)and Western Blot(WB)were used to detect the expression of HSPA5 and GPX4 in cervical cancer and paracancerous tissues.The relationship between HSPA5 and overall survival rate for all patients with cervical squamous cell carcinoma and endocervical adenocarcinoma(CESC)was analyzed based on TCGA database.Human cervical cancer HeLa and SiHa cell lines were cultured in vitro,and infected with shHSPA5 or shCtrl lentivirus to construct a stable strain of HSPA5 knockdown.qRT-PCR and WB were used to detected the expression of ferroptosis-related protein GPX4 after HSPA5 knockdown.CCK-8 and EdU cell proliferation assay,lactate dehydrogenase(LDH)assay,glutathione(GSH)kit,malondialdehyde(MDA)kit and flow cytometry analysis were used to determined cell proliferation,LDH,GSH and MDA content,cell apoptosis and reactive oxygen species(ROS)with ferroptosis inhibitor Ferrostatin-1(Fer-1)or ferroptosis activator Erastin treatment.Results:Compared with paracancerous tissue,the expression of HSPA5 and GPX4 in cervical cancer tissue was significantly higher.High HSPA5 level was consistent with poor prognosis.Knockdown of HSPA5 in HeLa and SiHa cells inhibited cell proliferation and increased cell apoptosis,meanwhile increased the levels of ROS,MDA and LDH in cells.Both mRNA and protein expression levels of GPX4 were reduced after HSPA5 knockdown,and the content of GSH in cells decreased.Treatment of Fer-1,an ferroptosis inhibitor,reversed the increased of ROS and LDH content induced by HSPA5 knockdown and inhibited cell apoptosis.In contrast,treatment of Erastin,an ferroptosis inducer,increased cell apoptosis and inhibitd cell proliferation cooperatively with HSPA5 knockdown.Conclusion:The results indicate that HSPA5 level is upregulated in cervical cancer tissues and HSPA5 knockdown inhibits proliferation and promotes apoptosis of cervical cancer cells through activat

关 键 词：HSPA5 铁死亡 宫颈癌 增殖 凋亡 

分 类 号：R737.33[医药卫生—肿瘤]