TXNIP/NLRP3通路在乳腺癌发生发展中的作用机制研究

Research on mechanism of action of TXNIP/NLRP3 pathway in the occurrence and development of breast cancer

目的探讨硫氧还蛋白结合蛋白(TXNIP)/核苷酸结合寡聚化结构域样受体3(NLRP3)通路在乳腺癌发生发展过程中的调控机制。方法选取2019年9月至2020年6月期间于笔者所在医院行手术切除的15例乳腺癌组织及其癌旁组织标本,通过免疫组化染色法检测其TXNIP及NLRP3蛋白的表达水平。同时选取3种乳腺癌细胞系(MDA-MB231、MCF-7和SKBR3)和正常乳腺上皮细胞系(HMEC),采用蛋白免疫印迹(Western blot)法检测各细胞系中TXNIP及NLRP3蛋白的相对表达水平。另取MDA-MB231乳腺癌细胞系将其分为6组,即空白对照组(正常培养不做任何处理)、TXNIP过表达组(Ad-TXNIP组,转染携带TXNIP过表达序列的腺病毒载体)、Ad-TXNIP阴性对照组(Ad-eGFP1组,转染不含TXNIP过表达序列的腺病毒空载体)、NLRP3过表达组(Ad-NLRP3组,转染含NLRP3过表达序列的腺病毒载体)、TXNIP和NLRP3过表达共转染组(Ad-TXNIP+Ad-NLRP3组,共转染携带TXNIP和NLRP3过表达序列的腺病毒载体)、TXNIP过表达和Ad-NLRP3阴性对照(Ad-eGFP2)共转染组(Ad-TXNIP+Ad-eGFP2组,共转染携带TXNIP过表达序列和不含NLRP3过表达序列的腺病毒空载体),各组细胞转染并培养24 h后,采用CCK-8法检测MDA-MB231细胞的增殖活性;采用Transwell小室法检测MDA-MB231细胞的迁移、侵袭能力;开展裸鼠成瘤实验并检测MDA-MB231细胞在动物体内的成瘤能力;采用Western blot法检测MDA-MB231细胞中TXNIP、NLRP3、增殖标志蛋白Ki-67、凋亡蛋白caspase-1、血管内皮生长因子(VEGF)、NLRP3下游蛋白白细胞介素(IL)-1β、IL-18以及caspase-1前体蛋白(pro-caspase-1)表达情况。结果与癌旁组织相比,乳腺癌组织中TXNIP蛋白相对表达水平降低(P<0.05),NLRP3蛋白相对表达水平升高(P<0.05)。与HMEC细胞系相比,MDA-MB231、MCF-7和SKBR3乳腺癌细胞系中TXNIP蛋白相对表达水平降低(P<0.05),NLRP3蛋白相对表达水平升高(P<0.05)。与空白对照组相比,Ad-TXNIP组及Ad-NLRP3组MDA-MB231细胞中TXNIP、NLRP3、IL-1β、IL-18、pro-caspase-1和caspase-1蛋白相对表达量升高(P<0.05),Ki-67和VEGF蛋白相对表达量,MDA-MB231细胞增殖活性、侵袭能力、迁移能力和动物体内瘤体质量均降低(P<0.05)。与Ad-TXNIP组及Ad-NLRP3组相比,Ad-TXNIP+Ad-NLRP3组MDA-MB231细胞中TXNIP、NLRP3、IL-1β、IL-18、pro-caspase-1和caspase-1蛋白相对表达量进一步升高(P<0.05),Ki-67和VEGF蛋白相对表达量,MDA-MB231细胞增殖活性、侵袭能力、迁移能力和动物体内瘤体质量进一步降低(P<0.05)。结论在乳腺癌组织及乳腺癌细胞系中,TXNIP蛋白呈低表达,NLRP3蛋白呈高表达;二者可相互作用,促进细胞焦亡,进而抑制乳腺癌细胞的增殖、侵袭与迁移。

Objective To investigate the regulatory mechanism of thioredoxin binding protein(TXNIP)/nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)pathway in the occurrence and development of breast cancer.Methods The resected 15 cases of breast cancer tissues and their adjacent tissues in our hospital from September 2019 to June 2020 were selected,and the immunohistochemistry was used to detect the expression levels of TXNIP and NLRP3 in breast cancer and its adjacent tissues.Three kinds of breast cancer cell lines(MDA-MB231,MCF-7 and SKBR3)and normal breast epithelial cell line(HMEC)were collected.Western blot was used to detect the relative expression levels of TXNIP and NLRP3 in three kinds of breast cancer cell lines and HMEC cell line.MDA-MB231 cancer cells were divided into blank control group(normal culture without any treatment),TXNIP overexpression group(Ad-TXNIP group,transfected with adenovirus vector carrying TXNIP overexpression sequence),Ad-TXNIP negative control group(Ad-eGFP1 group,transfected of empty adenovirus vector without TXNIP overexpression sequence),NLRP3 overexpression group(Ad-NLRP3 group,transfected with adenovirus vector containing NLRP3 overexpression sequence),TXNIP and NLRP3 overexpression co-transfection group(Ad-TXNIP+Ad-NLRP3 group,co-transfection of adenovirus vector carrying TXNIP and NLRP3 overexpression sequence),TXNIP overexpression and Ad-NLRP3 negative control(Ad-eGFP2)co-transfection group(Ad-TXNIP+Ad-eGFP2 group,co-transfection of adenovirus vector carrying TXNIP overexpression sequence and empty adenovirus without NLRP3 overexpression sequence).After 24 hours of transfection and culture,CCK-8 method was used to detect the MDA-MB231 cells proliferation.Transwell chamber method was used to detect MDA-MB231 cells migration and invasion.Nude mice tumorigenicity test was used to detect the tumorigenicity of the MDA-MB231 cells in vivo.Western blot was used to detect the expressions of TXNIP,NLRP3,proliferation marker protein(Ki-67),caspase-1,vascular endothelial growth factor(VEGF),interleukin(IL)-1β,IL-18 and caspase-1 precursor protein(pro-caspase-1)in the MDA-MB231 cells.Results Compared with the adjacent tissues,the relative expression level of TXNIP decreased(P<0.05)and the relative expression level of NLRP3 increased(P<0.05)in breast cancer tissues.Compared with normal breast epithelial cell line(HMEC cell line),the relative expression levels of TXNIP in MDA-MB231,MCF-7 and SKBR3 breast cancer cell lines were decreased(P<0.05),and the relative expression levels of NLRP3 were increased(P<0.05).Compared with the blank control group,the relative expression levels of TXNIP,NLRP3,IL-1β,IL-18,pro-caspase-1 and caspase-1 were increased(P<0.05),the relative expression levels of Ki-67 and VEGF,the proliferation activity,invasion and migration ability of MDA-MB231 cells and tumor weight were decreased(P<0.05)in the Ad-TXNIP group and the Ad-NLRP3 group.Compared with the Ad-TXNIP group and the Ad-NLRP3 group,the relative expression levels of TXNIP,NLRP3,IL-1β,IL-18,pro-caspase-1 and caspase-1 were further increased(P<0.05),the relative expression levels of Ki-67 and VEGF,the proliferation activity,invasion and migration ability of MDA-MB231 cells and tumor weight were further decreased(P<0.05)in the Ad-TXNIP+Ad-NLRP3 group.Conclusions In breast cancer tissues and breast cancer cell lines,TXNIP is low expression and NLRP3 is high expression.They can interact with each other to promote pyroptosis and inhibit the proliferation,invasion and migration of breast cancer cells.