TIPE通过调控LDHA表达影响三阴性乳腺癌糖代谢重编程机制研究

doi:10.19401/j.cnki.1007-3639.2025.04.006

摘要/Abstract

摘要：

背景与目的：肿瘤坏死因子-α诱导蛋白8（tumor necrosis factor alpha-induced protein 8，TNFAIP8，TIPE）在多种恶性肿瘤中发挥重要的调控作用，但其在三阴性乳腺癌（triple-negative breast cancer，TNBC）代谢重编程中的分子机制尚未阐明。本研究旨在揭示TIPE通过调控糖酵解关键酶乳酸脱氢酶A（lactate dehydrogenase A，LDHA）表达影响TNBC细胞增殖和糖代谢重编程的作用机制，为TNBC的临床治疗提供新的分子靶点。方法：采用慢病毒载体系统在MDA-MB-231细胞中建立TIPE-shRNA稳定转染株，通过嘌呤霉素筛选获得稳定细胞系，利用转录组测序分析TIPE对TNBC细胞糖代谢重编程通路的影响，通过SeaHorse能量代谢分析仪检测细胞外酸化率（extracellular acidification rate，ECAR），并结合乳酸检测等实验评估TIPE对TNBC细胞糖酵解能力的影响；采用Co-IP/MS技术筛选TIPE相互作用蛋白，在HEK-293T细胞中共转染TIPE-Myc和LDHA-Flag质粒，进一步验证TIPE与LDHA蛋白存在相互作用；通过CHX追踪实验和泛素化分析检测TIPE对LDHA蛋白稳定性的影响；通过细胞计数试剂盒（cell counting kit-8，CCK-8）CCK-8和ECAR实验、裸鼠移植瘤模型（伦理批号：202212007）结合免疫组织化学检测进一步验证TIPE介导LDHA影响TNBC细胞增殖与糖代谢重编程过程。结果：TIPE促进了TNBC细胞糖代谢重编程，实验数据显示，TIPE敲除显著抑制了TNBC细胞的糖酵解能力、糖酵解容量及乳酸生成量（P <0.001）；免疫共沉淀实验证实TIPE与糖酵解关键酶LDHA存在直接结合，进一步机制研究表明TIPE通过泛素化-蛋白酶体途径延缓LDHA蛋白降解过程，但对其mRNA表达水平无显著影响；细胞实验证实TIPE通过调控LDHA显著促进TNBC细胞增殖（P<0.001）和糖酵解活性（P<0.001），在裸鼠移植瘤模型中TIPE敲除组肿瘤体积显著缩小（P<0.05）、重量减轻（P<0.01），肿瘤组织免疫组织化学分析显示TIPE与LDHA表达呈显著正相关。结论：TIPE通过抑制LDHA泛素化水平降解促进TNBC细胞增殖与糖酵解能力。

关键词: 肿瘤坏死因子-α诱导蛋白8, 三阴性乳腺癌, 糖代谢重编程, 乳酸脱氢酶A, 泛素化

Abstract:

Background and purpose: Tumor necrosis factor alpha-induced protein 8 (TNFAIP8), also called TIPE, plays critical regulatory roles in various malignancies, yet its molecular mechanisms in metabolic reprogramming of triple-negative breast cancer (TNBC) remain elusive. This study aimed to elucidate how TIPE regulates the expression of the glycolytic key enzyme lactate dehydrogenase A to influence TNBC cell proliferation and glycolytic reprogramming, thereby providing potential molecular targets for TNBC therapy. Methods: Stable TIPE-knockdown MDA-MB-231 cell lines were established using a lentiviral shRNA system and selected with puromycin. Transcriptome sequencing was used to analyze TIPE's impact on TNBC glycolytic pathways. Extracellular acidification rate (ECAR) was measured using the Seahorse XF Analyzer, complemented by lactate production assays to evaluate glycolytic capacity. Co-IP/MS was carried out to identify TIPE-interacting proteins, with subsequent validation of TIPE-LDHA interaction through co-transfection of TIPE-Myc and LDHA-Flag plasmids in HEK-293T cells. Protein stability was assessed via cycloheximide (CHX) chase and ubiquitination assays. The cell counting kit-8 (CCK-8) assay and animal experiments (Approval Number for Animal Ethics: 202212007) were conducted to investigate how TIPE affects the proliferation and glucometabolic reprogramming of TNBC by mediating LDHA. Results: TIPE promoted glycolytic metabolic reprogramming in TNBC. Knockdown of TIPE significantly inhibited TNBC glycolytic activity and glycolytic capacity (P<0.001). TIPE interacted with the key glycolytic enzyme LDHA and suppressed its degradation rate through a ubiquitination-dependent mechanism. Cellular experiments demonstrated that TIPE mediated LDHA to enhance TNBC cell proliferation (P<0.001) and glycolytic activity (P<0.001). Animal studies confirmed that TIPE knockdown significantly suppressed tumor volume (P<0.05) and weight (P<0.01), with a positive correlation between TIPE and LDHA expression levels in tumor tissues. Conclusion: TIPE enhances TNBC cell proliferation and glycolytic capacity by inhibiting LDHA ubiquitination-mediated degradation.

Key words: TIPE, Triple-negative breast cancer, Glycometabolic reprogramming, Lactate dehydrogenase A, Ubiquitination

胡玮, 任晓朦, 王洋, 赵培庆, 曹凯. TIPE通过调控LDHA表达影响三阴性乳腺癌糖代谢重编程机制研究[J]. 中国癌症杂志, 2025, 35(4): 386-393.

HU Wei, REN Xiaomeng, WANG Yang, ZHAO Peiqing, CAO Kai. TIPE regulates glucometabolic reprogramming by modulating LDHA expression in triple-negative breast cancer[J]. China Oncology, 2025, 35(4): 386-393.

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