过氧化还原蛋白1的K197位点乳酸化修饰促进胶质母细胞瘤细胞的增殖和迁移  

Lactylation at K197 site of peroxiredoxin 1 promotes proliferation and migration of glioblastoma cells

作　　者：段国良 韩庆亮 范仕兵 DUAN Guoliang;HAN Qingliang;FAN Shibing(School of Medicine,Chongqing University,Chongqing 400000,China;Department of Neurosurgery,Chongqing Univer-sity Three Gorges Hospital,Chongqing 404000,China)

机构地区：[1]重庆大学医学院,重庆400000 [2]重庆大学附属三峡医院神经外科,重庆404000

出　　处：《中国病理生理杂志》2025年第2期219-229,共11页Chinese Journal of Pathophysiology

基　　金：中央高校前沿科学与颠覆性卡脖子技术研究子项医工融合项目(No.2021CDJYGRH-007);重庆市科技局自然面上项目(No.cstc2021jcyj-msxmX0999);重庆大学院级科研项目基础研究重点项目(No.2022YJKYXM-003)。

摘　　要：目的:探讨过氧化还原蛋白1(peroxiredoxin 1,PRDX1)的K197位点乳酸化修饰对胶质母细胞瘤细胞增殖和迁移的影响。方法:(1)通过免疫荧光和乳酸化泛抗体技术,对比胶质母细胞瘤组织与癌旁组织间PRDX1乳酸化修饰水平的差异,并结合高通量质谱与修饰组学分析,筛选出PRDX1蛋白及其K197位点作为研究焦点。(2)用乳酸(5、10和15 mmol/L)、葡萄糖(5、10和25 mmol/L)或糖酵解抑制剂2-脱氧-D-葡萄糖(2-deoxy-D-glucose,2-DG;1、5、10和15 mmol/L)处理人胶质母细胞瘤U87MG和LN229细胞,用EdU增殖染色法检测细胞增殖。取U87MG、LN229细胞和神经胶质细胞,用免疫沉淀和Western blot法检测PRDX1的表达。对10 mmol/L乳酸组和未加乳酸组细胞用免疫沉淀和Western blot法检测PRDX1表达及乳酸化修饰水平。(3)构建乳酸脱氢酶A(lactate dehydrogenase A,LDHA)siRNA(si-LDHA)质粒和阴性对照质粒si-Con,转染U87MG和LN229细胞,免疫沉淀和Western blot法分别检测PRDX1乳酸化修饰水平。(4)构建PRDX1 shRNA(sh-PRDX1)质粒和阴性对照质粒sh-Con,转染U87MG和LN229细胞,Western blot法检测PRDX1的表达。(5)构建PRDX1 K197R(突变型)质粒、PRDX1 WT(野生型)质粒和sh-PRDX1质粒,转染U87MG和LN229细胞,用免疫沉淀和Western blot法检测PRDX1表达与乳酸化修饰水平。取PRDX1 K197R和PRDX1 WT转染U87MG和LN229细胞后,CCK-8法和EdU法检测细胞活力和增殖,Transwell法检测细胞迁移。(6)构建裸鼠成瘤模型,将PRDX1基因K197R突变及未处理的LN229细胞进行裸鼠体内成瘤实验,每组6只裸鼠,18 d后处死裸鼠,取肿瘤组织。用HE染色法观察组织变化,免疫荧光法检测乳酸化修饰水平,免疫组化法检查肿瘤组织内增殖标志物Ki67的表达。结果:胶质母细胞瘤组织中的PRDX1乳酸化修饰水平显著高于癌旁组织(P<0.05)。在细胞实验中,乳酸和葡萄糖的添加显著促进了胶质母细胞瘤的增殖和迁移,并增加了PRDX1的乳酸化修饰水平(P<0.05);�AIM:To investigate the effects of lactylation at the K197 site of peroxiredoxin 1(PRDX1)on the proliferation and migration of glioblastoma cells.METHODS:(1)Immunofluorescence and lactylation pan-antibody techniques were adopted to compare the differences in PRDX1 lactylation modification level between glioblastoma tissues and adjacent normal tissues.High-throughput mass spectrometry and modificomics analysis were utilized to select PRDX1 protein and its K197 site as the focus.(2)Cell experiments were conducted using lactate(5,10 and 15 mmol/L),glu-cose(5,10 and 25 mmol/L)and glycolysis inhibitor 2-deoxy-D-glucose(2-DG;1,5,10 and 15 mmol/L)to treat human glioblastoma U87MG and LN229 cells.Cell proliferation was detected by EdU proliferation staining,and PRDX1 expres-sion was detected in U87MG,LN229 and glial cells via immunoprecipitation and Western blot.The PRDX1 expression and lactylation levels were further examined in 10 mmol/L lactic acid-treated and untreated cells using immunoprecipita-tion and Western blot.(3)The U87MG and LN229 cells were transfected with constructed lactate dehydrogenase A(LDHA)siRNA(si-LDHA)plasmids and negative control(si-Con)plasmids,and the lactylation level of PRDX1 was as-sessed by immunoprecipitation and Western blot.(4)Similarly,the U87MG and LN229 cells were transfected with PRDX1 shRNA(sh-PRDX1)plasmids and negative control(sh-Con)plasmids,and PRDX1 expression was determined by Western blot.(5)The PRDX1 K197R mutant and PRDX1 wild-type(WT)plasmids were constructed and transfected into U87MG and LN229 cells.The PRDX1 expression and lactylation levels were determined by immunoprecipitation and Western blot.The CCK-8 and EdU assays were used to measure cell viability and proliferation,and Transwell assay was performed to assess the migration of U87MG and LN229 cells transfected with PRDX1 K197R mutant and PRDX1 WT plasmids.(6)A tumor formation model in nude mice was established.The LN229 cells with or without PRDX1 K197R mutation were used in the tumor formation experiment with 6

关 键 词：胶质母细胞瘤 过氧化还原蛋白1 乳酸化 点突变 细胞增殖 细胞迁移 

分 类 号：R739.41[医药卫生—肿瘤] R363[医药卫生—临床医学] Q279[生物学—细胞生物学]