METTL3通过m6A调控GBAP1经PI3K/AKT通路促膀胱癌进展的影响OA
METTL3 Promotes Bladder Cancer Progression via m6A-Modified LncRNA GBAP1 Through PI3K/AKT Pathway
目的:旨在揭示 METTL3 通过 m6A 修饰调控 LncRNA GBAP1 表达,进而激活 PI3K/AKT通路促进膀胱癌进展的分子机制.方法:qRT-PCR 检测人正常尿上皮细胞系 SV-HUC-1 和膀胱癌细胞系(T24、UM-UC-3、J82 和5637)中 METTL3 和 GBAP1 表达水平.将膀胱癌细胞 T24 随机分成9 组:pcDNA NC 组、pcDNA METTL3 组、sh-NC 组、sh-METTL3 组、sh-METTL3+oe-NC 组、sh-METTL3+oe-GBAP1 组、si-NC 组、si-GBAP1 组和 si-GBAP1+740Y-P 组.采用甲基化 RNA 免疫共沉淀和 RNA 免疫沉淀分析 METTL3 介导 GBAP 的 m6A 修饰水平.平板克隆实验、Transwell 实验和细胞划痕实验检测细胞侵袭和迁移水平.检测细胞内 Cu+水平.蛋白免疫印迹检测铜死亡和 PI3K/AKT 轴相关蛋白表达水平的影响.裸鼠移植瘤检测移植瘤体积和质量.结果:与人正常尿上皮细胞系 SV-HUC-1 相比,METTL3 和 GBAP1 在膀胱癌细胞系(T24、UM-UC-3、J82 和5637)中的表达显著上调(P<0.05),在 T24中差异表达最明显.甲基化 RNA 免疫共沉淀和 RNA 免疫沉淀分析显示 METTL3 抗体促进 GBAP1 m6A甲基化水平显著富集且 GBAP1 表达水平显著增加(P<0.05).与 sh-NC 组相比,sh-METTL3 组的GBAP1 表达、平板克隆数、侵袭细胞数、细胞迁移率、p-PI3K/PI3K 和 p-AKT/AKT 表达水平、裸鼠移植瘤体积和质量显著降低(P<0.05),Cu+水平、FDX1、LIAS、LIPT1 和 DLAT 蛋白表达水平显著增加(P<0.05).与 sh-METTL3+oe-NC 组相比,sh-METTL3+oe-GBAP1 组的 GBAP1 表达、平板克隆数、侵袭细胞数、细胞迁移率、p-PI3K/PI3K 和 p-AKT/AKT 表达水平、裸鼠移植瘤体积和质量显著增加(P<0.05),Cu+水平、FDX1、LIAS、LIPT1 和 DLAT 蛋白表达水平显著降低(P<0.05).与 si-NC 组相比,si-GBAP1组的 p-PI3K/PI3K、p-AKT/AKT 表达水平、平板克隆数、侵袭细胞数和细胞迁移率显著降低(P<0.05),Cu+水平、FDX1、LIAS、LIPT1 和 DLAT 蛋白表达水平显著增加(P<0.05).与 si-GBAP1 组相比,si-GBAP1+740Y-P 组的 p-PI3K/PI3K、p-AKT/AKT 表达水平、平板克隆数、侵袭细胞数和细胞迁移率显著增加(P<0.05),Cu+水平、FDX1、LIAS、LIPT1 和 DLAT 蛋白表达水平显著降低(P<0.05).结论:在膀胱癌细胞中,METTL3 和 GBAP1 表达显著上调.METTL3 通过 m6A 甲基化修饰调控 LncRNA GBAP1 的表达,进而经由 PI3K/AKT 通路抑制膀胱癌细胞铜死亡,促进其恶性进展.
Objective:To reveal the molecular mechanism by which METTL3 regulates LncRNA GBAP1 expression through m6A modification,thereby activating the PI3K/AKT pathway and promoting bladder cancer progression.Methods:qRT-PCR was used to detect the expression levels of METTL3 and GBAP1 in the nor-mal human uroepithelial cell line SV-HUC-1 and bladder cancer cell lines(T24,UM-UC-3,J82,and 5637).T24 cells were divided into nine groups:pcDNA NC,pcDNA METTL3,sh-NC,sh-METTL3,sh-METTL3+oe-NC,sh-METTL3+oe-GBAP1,si-NC,si-GBAP1,and si-GBAP1+740Y-P.Methylated RNA immunoprecipitation(MeRIP)and RNA immunoprecipitation(RIP)assays were performed to assess METTL3-mediated m6A modification levels of GBAP1.Colony formation,Transwell,and wound healing as-says were used to evaluate cell invasion and migration.Intracellular Cu+levels were measured.Western blot-ting was performed to detect the expression of cuproptosis-related proteins and PI3K/AKT pathway compo-nents.A nude mouse xenograft model was used to assess tumor volume and weight.Results:Compared with SV-HUC-1 cells,both METTL3 and GBAP1 were significantly upregulated in bladder cancer cell lines(T24,UM-UC-3,J82,and 5637)(P<0.05),with the most pronounced differences observed in T24 cells.MeRIP and RIP assays showed that METTL3 antibody significantly enriched the m6A methylation level of GBAP1 and increased GBAP1 expression(P<0.05).Compared with the sh-NC group,the sh-METTL3 group showed significantly reduced GBAP1 expression,colony number,invading cell number,cell migration rate,p-PI3K/PI3K and p-AKT/AKT ratios,as well as xenograft tumor volume and weight(P<0.05),while Cu+levels and the protein levels of FDX1,LIAS,LIPT1,and DLAT were significantly increased(P<0.05).Compared with the sh-METTL3+oe-NC group,the sh-METTL3+oe-GBAP1 group exhibited significantly in-creased GBAP1 expression,colony number,invading cell number,cell migration rate,p-PI3K/PI3K and p-AKT/AKT ratios,and xenograft tumor volume and weight(P<0.05),along with decreased Cu+levels and FDX1,LIAS,LIPT1,and DLAT protein levels(P<0.05).Compared with the si-NC group,the si-GBAP1 group had significantly lower p-PI3K/PI3K and p-AKT/AKT ratios,colony number,invading cell number,and cell migration rate(P<0.05),while Cu+levels and FDX1,LIAS,LIPT1,and DLAT protein levels were significantly higher(P<0.05).Compared with the si-GBAP1 group,the si-GBAP1+740Y-P group showed significantly increased p-PI3K/PI3K and p-AKT/AKT ratios,colony number,invading cell number,and cell migration rate(P<0.05),and decreased Cu+levels and FDX1,LIAS,LIPT1,and DLAT protein levels(P<0.05).Conclusion:METTL3 and GBAP1 are significantly upregulated in bladder cancer cells.MET-TL3 regulates LncRNA GBAP1 expression via m6A methylation,thereby inhibiting cuproptosis and promoting malignant progression of bladder cancer through the PI3K/AKT pathway.
吴文婧;李淑洁;徐加佳;关文芳;尹倩雯;孙晓敏;陈葳
西安交通大学第一附属医院检验科,陕西 西安 710061西安交通大学第一附属医院检验科,陕西 西安 710061西安交通大学第一附属医院检验科,陕西 西安 710061西安交通大学第一附属医院检验科,陕西 西安 710061西安交通大学第一附属医院检验科,陕西 西安 710061西安交通大学第一附属医院检验科,陕西 西安 710061西安交通大学第一附属医院检验科,陕西 西安 710061
膀胱癌METTL3LncRNA GBAP1m6A铜死亡
Bladder cancerMETTL3LncRNA GBAP1m6ACuproptosis
《河北医学》 2026 (5)
705-713,9
陕西省自然科学基金(编号:2023-JC-YB-666)
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