摘要
目的探讨调节蛋白2(SIRT2)对三阴性乳腺癌(TNBC)细胞增殖和有氧糖酵解的调控作用。方法采用Kaplan-Meier Plotter评估SIRT2对TNBC预后的影响;RT-qPCR及Western blot检测MCF-10A人乳腺上皮细胞与MDA-MB-231三阴性乳腺癌细胞中SIRT2、丙酮酸激酶M2型同工酶(PKM2)、乳酸脱氢酶A(LDHA)的表达;免疫组化染色比较纤维腺病与TNBC组织中SIRT2、BCL2关联的X蛋白(Bax)及B淋巴细胞瘤-2(Bcl-2)蛋白的表达;慢病毒载体构建SIRT2表达的TNBC细胞稳转株,通过CCK-8、Edu、Transwell检测SIRT2对TNBC细胞的增殖、迁移和侵袭能力的影响,Western blot检测细胞的凋亡情况;加利福尼亚大学圣塔克鲁兹分校基因组数据库(UCSC)和Spearman分析PKM2、LDHA表达及与糖酵解相关性;葡萄糖氧化酶-比色法检测SIRT2对糖酵解相关酶通路调控PKM2和LDHA的影响。结果Kaplan-Meier生存分析结果显示,TNBC的SIRT2高表达组的无复发生存期(RFS)更短(P<0.05);RT-qPCR和Western blot发现SIRT2在MDA-MB-231细胞中高表达(P<0.05);免疫组织化学染色显示SIRT2、Bax在TNBC组织中高表达,而Bcl-2在TNBC中低表达(P<0.05);SIRT2表达促进TNBC细胞增殖,增强TNBC细胞的迁移和侵袭能力,敲低SIRT2可促进TNBC细胞促凋亡蛋白Bax、pro Caspase-3、cleaved Caspase-9的表达水平,抑制了抑凋亡蛋白Bcl-2的表达水平(P<0.05);生物信息学分析显示PKM2和LDHA在TNBC中高表达,且相关性分析显示SIRT2表达与糖酵解通路呈正相关(P<0.05);RT-qPCR和Western blot结果显示,与MCF-10A相比,PKM2和LDHA在MDA-MB-231细胞中高表达(P<0.05);敲低SIRT2可减少TNBC细胞葡萄糖消耗和乳酸生成,下调PKM2和LDHA蛋白的表达水平(P<0.05)。结论SIRT2促进TNBC细胞有氧糖酵解及细胞生长增殖,其机制可能与SIRT2调控以及PKM2、LDHA表达有关。
Objective To investigate the regulatory role of Sirtuin 2(SIRT2)in the proliferation and aerobic glycolysis of triple-negative breast cancer(TNBC)cells.Methods Kaplan-Meier Plotter was used to assess the impact of SIRT2 on TNBC prognosis.RT-qPCR and Western blot were employed to detect the expression of SIRT2,pyruvate kinase M2 isozyme(PKM2),and lactate dehydrogenase A(LDHA)in MCF-10A human mammary epithelial cells and MDA-MB-231 TNBC cells.Immunohistochemical staining was performed to compare the expression of SIRT2,BCL2-associated X protein(Bax),and B-cell lymphoma-2(Bcl-2)in fibroadenosis and TNBC tissues.Lentiviral vectors were used to construct stable TNBC cell lines with altered SIRT2 expression.The effects of SIRT2 on TNBC cell proliferation,migration,and invasion were evaluated using CCK-8,EdU,and Transwell assays,while Western blot was used to assess apoptosis.The UCSC Genome Database and Spearman analysis were applied to examine PKM2 and LDHA expression and their correlation with glycolysis.Glucose oxidase-colorimetric assay was used to investigate the regulatory effects of SIRT2 on PKM2 and LDHA through the glycolytic enzyme pathway.Results Kaplan-Meier survival analysis showed that TNBC patients with high SIRT2 expression had shorter relapse-free survival(RFS),the difference was statistically significant(P<0.05).RT-qPCR and Western blot revealed high expression of SIRT2 in MDA-MB-231 cells(P<0.05).Immunohistochemical staining demonstrated high expression of SIRT2 and Bax in TNBC tissues,while Bcl-2 was expressed at lower levels(P<0.05).Overexpression of SIRT2 promoted TNBC cell proliferation and enhanced migration and invasion capabilities,whereas SIRT2 knockdown upregulated the expression of pro-apoptotic proteins Bax,pro-Caspase-3,and cleaved Caspase-9 while downregulating the anti-apoptotic protein Bcl-2(P<0.05).Bioinformatics analysis indicated high expression of PKM2 and LDHA in TNBC,with correlation analysis showing a positive association between SIRT2 expression and the glycolysis pathway(P<0.05).RT-qPCR and Western blot results showed that PKM2 and LDHA were highly expressed in MDA-MB-231 cells compared with MCF-10A cells(P<0.05).SIRT2 knockdown reduced glucose consumption and lactate production in TNBC cells and downregulated PKM2 and LDHA protein expression levels(P<0.05).Conclusion SIRT2 promotes aerobic glycolysis,growth,and proliferation in TNBC cells,and its mechanism may be related to the regulation of PKM2 and LDHA expression by SIRT2.
作者
杨冰清
杨宇石
徐澍
YANG Bingqing;YANG Yushi;XU Shu(Department of Pathology,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou,China)
出处
《贵州医科大学学报》
2026年第2期168-176,共9页
Journal of Guizhou Medical University
基金
国家自然科学基金培育项目(I-2020-18)
贵州省科技厅黔科合成果项目(LC〔2023〕030)。
