α2-巨球蛋白表达下调激活TGF-β/SMADs信号通路诱导卵巢癌顺铂耐药  

Down-regulation ofα2-macroglobulin expression activates TGF-β/SMADs signaling pathway to induce cisplatin resistance in ovarian cancer

作　　者：陆婷婷 石丽君 刘萤照 梁惠婷 王琪[1,3] LU Tingting;SHI Lijun;LIU Yingzhao;LIANG Huiting;WANG Qi(Department of Experimental Research,Guangxi Medical University Cancer Hospital;Department of Oncology,The First Affiliated Hospital of Guangxi Medical University;Key Laboratory of Early Prevention and Treatment for Regional High Frequency Tumor(Guangxi Medical University),Ministry of Education,Nanning 530021,China)

机构地区：[1]广西医科大学附属肿瘤医院实验研究部,南宁530021 [2]广西医科大学第一附属医院肿瘤科 [3]区域性高发肿瘤早期防治研究教育部重点实验室(广西医科大学)

出　　处：《中国癌症防治杂志》2024年第6期707-714,共8页CHINESE JOURNAL OF ONCOLOGY PREVENTION AND TREATMENT

基　　金：国家自然科学基金青年科学基金项目(82102962);国家自然科学基金地区科学基金项目(81360341)。

摘　　要：目的探讨卵巢癌患者体内α2-巨球蛋白(alpha-2-macroglobulin,A2M)表达水平与患者预后及铂类耐药之间的关系,并分析A2M对TGF-β/SMADs信号通路的调控以及对顺铂(Cisplatin,DDP)诱导肿瘤细胞凋亡的影响。方法采用Western blot检测经DDP治疗裸鼠卵巢癌异种皮下移植瘤模型移植瘤中A2M表达并分析其与DDP耐药间的关系;免疫组织化学法检测129例卵巢癌组织中A2M的表达及分析其与患者预后的关系;ELISA检测103例卵巢癌血清样本中A2M含量及分析其与DDP耐药的关系。在耐药的卵巢癌细胞SKOV3/DDPⅡ中外源添加0、0.5、1.0、2.0、4.0μmol/L A2M蛋白,ELASA检测共培养体系中A2M含量并分析其与细胞因子间的关系,流式细胞术检测DDP耐药卵巢癌细胞凋亡情况。利用RNA干扰技术敲低敏感细胞SKOV3中A2M基因的表达,CCK-8实验检测经外源添加和敲低A2M两种处理后各组细胞活性并计算DDP的半数抑制浓度(half maximal inhibitory concentration,IC50);Western blot检测两种处理后各组细胞中TGF-β/SMADs信号传导的关键节点TGF-β1、TGFβR2、SMAD2/3及其磷酸化表达,以及细胞凋亡相关蛋白BCL-2和PARP1的表达。采用裸鼠卵巢癌异种皮下移植瘤模型,比较DDP治疗对A2M基因敲低的SKOV3细胞和对照细胞成瘤体积的影响。结果耐药小鼠移植瘤中的A2M蛋白表达随DDP治疗次数增加而下降,但PARP1蛋白表达则上调。A2M蛋白表达与卵巢癌患者总生存期和无进展生存期均呈正相关(均P<0.05),铂耐药卵巢癌患者血清中A2M含量下降(P=0.031)。外源加入A2M蛋白可使TGF-β1、MMP1、MMP2、MMP7、MMP9细胞因子含量均较未加入A2M蛋白显著下降(均P<0.05),SKVO3/DDPⅡ细胞和A2780/DDPⅡ细胞总凋亡率上升和对DDP的IC50下降(P<0.0001),同时降低SKVO3/DDPⅡ细胞TGF-β1、PKCa的表达及下调TGFβR2、SAMD2/3的磷酸化水平,抑制TGF-β1-SMAD2/3复合物发挥转录因子作用,进而抑制凋亡相关蛋白BCL-2磷酸化及促�Objective To investigate the relationship between the expression levels of alpha-2-macroglobulin(A2M)in ovarian cancer patients and their prognosis and platinum resistance,and to analyze the regulation of A2M on the TGF-β/SMADs signaling pathway and its impact on the apoptosis of tumor cells induced by Cisplatin(DDP).Methods The Western blot was used to detect A2M expression in xenogeneic subcutaneous tumor models of ovarian cancer treated by DDP in nude mice,and the relationship between A2M expression and DDP resistance was analyzed.Immunohistochemistry was used to detect the expression of A2M in 129 ovarian cancer tissues and analyze its relationship with prognosis.ELISA was used to detect A2M content and analyze its relationship with DDP resistance in serum samples of 103 cases of ovarian cancer.The 0,0.5,1.0,2.0,4.0μmol/L A2M protein was added to drug-resistant ovarian cancer cells SKOV3/DDPⅡ,A2M content in co-culture system was detected by ELASA and analyzed its relationship with cytokines,and the apoptosis of DDP drugresistant ovarian cancer cells was detected by flow cytometry.The expression of A2M gene in sensitive cells SKOV3 was knocked down by the RNA interference technology,CCK-8 assay was used to detect the cell activity of each group after treated exogenous A2M and knocking down A2M,and the half maximal inhibitory concentration(IC50)of DDP was calculated;The Western blot was used to detect the key nodes of TGF-β/SMADs signal transduction,TGF-β1,TGFβR2,SMAD2/3,and their phosphorylated expression,and the expression of cell apoptosis-related proteins BCL-2 and PARP1.A xenograft model of ovarian cancer in nude mice was used to compare the effect of DDP treatment on the tumorigenesis volume of SKOV3 cells with A2M gene knockdown and control cells.Results The expression of A2M protein in xenografts of drug-resistant mice decreased with the increase of DDP treatment,whereas the expression of PARP1 protein was up-regulated.A2M protein expression was positively correlated with overall survival and pro

关 键 词：卵巢癌 Α2-巨球蛋白 顺铂 耐药 TGF-β/SMADs信号通路 

分 类 号：R737.31[医药卫生—肿瘤]