Mechanism of TPI-1 in Promoting the Proliferation and Invasion of Gastric Cancer Cells through Glycolytic Reprogramming
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摘要:
目的 从糖酵解重编程的角度探讨TPI-1干预胃癌进展可能的作用机制。 方法 构建TPI-1敲减、过表达胃癌细胞模型，将实验细胞分为shNC、shTPI-1、oe-Ctrl、oe-TPI-1四组，CCK-8法检测不同组胃癌细胞活性；Transwell法检测不同组胃癌细胞侵袭能力；糖酵解压力测试检测ECAR反映不同组胃癌细胞糖酵解产能的能力；通过Western blot 分析不同组细胞凋亡相关、侵袭相关、糖酵解相关特征标志物的表达量；免疫荧光和co-IP实验验证TPI-1与HK2共定位和结合的关系。 结果 与shNC组相比，shTPI-1组细胞增殖及侵袭能力减弱（P < 0.01），与oe-Ctrl组相比，oe-TPI-1组增殖和侵袭能力增强（P < 0.01）；在敲减TPI-1后，基础糖酵解能力及最大糖酵解能力均降低（P < 0.01）；shTPI-1组中，糖酵解关键酶HK2和PKM2的蛋白水平显著降低（P < 0.01），MMP-2、MMP-9、N-cadherin、Bcl-2、COX4I1显著下调（P < 0.01），NOX4、E-cadherin、Bax、cleaved caspase-3显著上调（P < 0.01），oe-TPI-1组中则呈现相反趋势；免疫荧光分析发现，存在TPI-1与HK2在细胞内共定位；免疫共沉淀初步证实TPI-1与HK2可能存在结合。 结论 TPI-1通过与HK2互作，持续激活糖酵解起始步骤，促进糖酵解重编程，从而促进胃癌进展。 Abstract:Objective To investigate the potential mechanism of TPI-1 intervention in gastric cancer progression from the perspective of glycolytic reprogramming. Methods TPI-1 knockdown and overexpression gastric cancer cell models were constructed. Experimental cells were divided into four groups: shNC, shTPI-1, oe-Ctrl, and oe-TPI-1. Cell viability was detected using the CCK-8 assay; invasive capacity was assessed using the Transwell assay; glycolytic capacity was measured using glycolytic stress test to detect ECAR reflecting the glycolytic capacity of different groups of gastric cancer cells; Western blot was used to analyze the expression levels of apoptosis-related, invasion-related, and glycolysis-related characteristic markers in different groups; immunofluorescence and co-IP experiments were performed to verify the colocalization and binding relationship between TPI-1 and HK2. Results Compared with the shNC group, the shTPI-1 group showed reduced cell proliferation and invasion capacity （P < 0.01）; compared with the oe-Ctrl group, the oe-TPI-1 group demonstrated enhanced proliferation and invasion capacity （P < 0.01）; after TPI-1 knockdown, both basal glycolytic capacity and maximum glycolytic capacity were decreased （P < 0.01）; in the shTPI-1 group, protein levels of glycolytic key enzymes HK2 and PKM2 were significantly reduced （P < 0.01）, and MMP-2, MMP-9, N-cadherin, Bcl-2, and COX4I1 were significantly downregulated （P < 0.01）, while NOX4, E-cadherin, Bax, and cleaved caspase-3 were significantly upregulated （P < 0.01）; the oe-TPI-1 group showed opposite trends; immunofluorescence analysis revealed colocalization of TPI-1 and HK2 within cells; co-immunoprecipitation preliminarily confirmed that TPI-1 and HK2 may interact. Conclusion TPI-1 promotes gastric cancer progression by interacting with HK2, continuously activating the initiation step of glycolysis, and promoting glycolysis reprogramming. -
Key words:
- TPI-1 /
- HK2 /
- Glycolysis /
- ECAR /
- Energy metabolism /
- Gastric cancer
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图 1 TPI-1在各细胞中表达情况及敲减和过表达验证
A：TPI-1在人正常胃黏膜细胞株GES-1和人胃癌细胞株SGC-7901、NCI-N87、MKN45中表达情况；B：TPI-1在人正常胃黏膜细胞株GES-1和人胃癌细胞株SGC-7901、NCI-N87、MKN45中相对表达含量柱状图；与GES-1组比较，**P < 0.01；C：在MKN45细胞中进行TPI- 1敲减以及在NCI-N87进行TPI-1过表达后TPI- 1的含量变化，验证敲减和过表达效率。
Figure 1. Expression of TPI-1 in various cells and verification of knockdown and overexpression
图 2 TPI-1对胃癌细胞侵袭的影响（200 μm，40×）
Figure 2. Effects of TPI-1 on gastric cancer cell invasion （200 μm，40 ×）
图 3 TPI-1对胃癌细胞相关蛋白的影响
A：HK2、PKM2、Bax、cleaved caspase-3、Bcl-2、MMP-2、MMP-9、N-cadherin、E-cadherin、NOX4和COX4I1蛋白与对照细胞相比，在shTPI-1细胞与oe-TPI-1细胞中的表达情况；B：蛋白表达含量的量化柱状图，与shCtrl组比较，**P < 0.01；***P < 0.001；与oe-Ctrl组比较，**P < 0.01；***P < 0.001。HK2：己糖激酶2；PKM2：丙酮酸激酶同工酶； MMP-2：金属蛋白酶-2；MMP-9：金属蛋白酶-9；E-cadherin：E型钙粘蛋白；N-cadherin：N型钙粘蛋白；Bcl-2：B淋巴细胞瘤-2；Bax：促凋亡蛋白；cleaved caspase-3：剪切的半胱天冬蛋白酶-3；NOX4: NADPH氧化酶4；COX4I1：细胞色素c氧化酶亚基。
Figure 3. Effects of TPI-1 on gastric cancer cell-related proteins
图 4 TPI-1对胃癌细胞能量代谢的影响
与shCtrl组比较，**P < 0.01；***P < 0.001。
Figure 4. Effects of TPI-1 on energy metabolism in gastric cancer cells
图 5 TPI-1和HK2在细胞内共定位
A：仅加二抗与DAPI，无一抗孵育，验证荧光信号特异性；B：正常孵育，在MKN45中的TPI-1与HK2的免疫荧光；C：在MKN45中分别以TPI-1和HK2的抗体作为一抗与磁珠偶联进行的免疫共沉淀；D：免疫共沉淀WB的量化柱状图；与IgG比较， ***P < 0.001。
Figure 5. Colocalization of TPI-1 and HK2 in cells
表 1 在MKN45细胞敲减TPI-1后增殖能力比较（$\bar x \pm s $，n = 3）
Table 1. Comparison of proliferation ability in MKN45 cells after TPI-1 knockdown （$\bar x \pm s $，n = 3）
组别 24 h 48 h 72 h shCtrl 0.48 ± 0.08 0.85 ± 0.05 1.16 ± 0.07 shTPI-1 0.43 ± 0.05 0.48 ± 0.06 0.85 ± 0.05 t 1.10 8.32 6.82 P 0.33 0.001** 0.002** **P < 0.01。 
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表 2 在N87细胞中过表达TPI-1后增殖能力比较（$\bar x \pm s $，n = 3）
Table 2. Comparison of proliferation ability after overexpressing TPI-1 in N87 cells （$\bar x \pm s $，n = 3）
组别 24 h 48 h 72 h oe-Ctrl 0.41 ± 0.01 0.50 ± 0.06 0.70 ± 0.04 oe-TPI-1 0.41 ± 0.06 0.48 ± 0.06 0.85 ± 0.04 t 0.19 0.29 5.22 P 0.86 0.79 0.006** **P < 0.01。
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表 3 TPI-1对胃癌细胞侵袭的影响（$\bar x \pm s $，n = 3）
Table 3. Effects of TPI-1 on gastric cancer cell invasion（$\bar x \pm s $，n = 3）
组别 细胞个数 shCtrl 86.30 ± 3.79 shTPI-1 27.00 ± 2.65 oe-Ctrl 28.70 ± 6.03 oe-TPI-1 74.30 ± 2.08 F 182.60 P < 0.001*** 与对照组比较，***P < 0.001。
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表 4 TPI-1对胃癌细胞能量代谢的影响（$\bar x \pm s $，n = 3）
Table 4. Effects of TPI-1 on energy metabolism in gastric cancer cells （$\bar x \pm s $，n = 3）
组别 shCtrl shTPI-1 t P 基础糖酵解ECAR（mpH/min） 77.70 ± 1.53 61.70 ± 1.53 12.83 < 0.001*** 最大糖酵解ECAR（mpH/min） 100.30 ± 2.50 82.70 ± 2.10 9.37 < 0.001*** 与shCtrl比较，***P < 0.001。
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[1] López M J, Carbajal J, Alfaro A L, et al. Characteristics of gastric cancer around the world[J]. Crit Rev Oncol Hematol, 2023, 181: 103841. doi: 10.1016/j.critrevonc.2022.103841 [2] Röcken C. Predictive biomarkers in gastric cancer[J]. J Cancer Res Clin Oncol, 2023, 149(1): 467-481. doi: 10.1007/s00432-022-04408-0 [3] Guan W L, He Y, Xu R H. Gastric cancer treatment: Recent progress and future perspectives[J]. J Hematol Oncol, 2023, 16(1): 57. doi: 10.1186/s13045-023-01451-3 [4] Sun L, Zhang H, Gao P. Metabolic reprogramming and epigenetic modifications on the path to cancer[J]. Protein Cell, 2022, 13(12): 877-919. doi: 10.1007/s13238-021-00846-7 [5] Zhu S, Guo Y, Zhang X, et al. Pyruvate kinase M2 (PKM2) in cancer and cancer therapeutics[J]. Cancer Lett, 2021, 503: 240-248. [6] Merkle S, Pretsch W. Characterization of triosephosphate isomerase mutants with reduced enzyme activity in Mus musculus[J]. Genetics, 1989, 123(4): 837-844. doi: 10.1093/genetics/123.4.837 [7] Grosso A R. Tooth hop variability in human and nonhuman bone: Effect on the estimation of saw blade TPI[J]. J Forensic Sci, 2022, 67(1): 102-111. doi: 10.1111/1556-4029.14897 [8] An Y, Cao C, Sun S, et al. SHP1 and its downstream p38/SP1/PI3K/YAP/Notch-1 signaling in trophoblast cells suppressed the progression of Preeclampsia via inhibiting proliferation of SMCs[J]. Sci Rep, 2025, 15(1): 16205. doi: 10.1038/s41598-025-00164-6 [9] 王冠. TPI1通过糖酵解与p53信号通路促进胃癌增殖、侵袭及细胞周期[D]. 兰州: 兰州大学, 2023. [10] Li Z, Li J, Li L, et al. Klotho enhances stability of chronic kidney disease atherosclerotic plaques by inhibiting GRK2/PLC-β-mediated endoplasmic reticulum stress in macrophages via modulation of the ROS/SHP1 pathway[J]. Sci Rep, 2024, 14(1): 32091. doi: 10.1038/s41598-024-83596-w [11] Rho H, Terry A R, Chronis C, et al. Hexokinase 2-mediated gene expression via histone lactylation is required for hepatic stellate cell activation and liver fibrosis[J]. Cell Metab, 2023, 35(8): 1406-1423. e8. [12] Bryan N, Raisch K P. Identification of a mitochondrial-binding site on the N-terminal end of hexokinase II[J]. Biosci Rep, 2015, 35(3): e00205. doi: 10.1042/BSR20150047 [13] Fontana F, Giannitti G, Marchesi S, et al. The PI3K/Akt pathway and glucose metabolism: A dangerous liaison in cancer[J]. Int J Biol Sci, 2024, 20(8): 3113-3125. doi: 10.7150/ijbs.89942 [14] Zhu S, Guo Y, Zhang X, et al. Pyruvate kinase M2 (PKM2) in cancer and cancer therapeutics[J]. Cancer Lett, 2021, 503: 240-248. [15] Liang L J, Yang F Y, Wang D, et al. CIP2A induces PKM2 tetramer formation and oxidative phosphorylation in non-small cell lung cancer[J]. Cell Discov, 2024, 10(1): 13. doi: 10.1038/s41421-023-00633-0 [16] Snider G W. Proteopedia entry: Triose phosphate isomerase[J]. Biochem Mol Biol Educ, 2011, 39(6): 464. doi: 10.1002/bmb.20550 [17] Pekel G, Ari F. Therapeutic targeting of cancer metabolism with triosephosphate isomerase[J]. Chem Biodivers, 2020, 17(5): e2000012. doi: 10.1002/cbdv.202000012 [18] Grüning N M, Du D, Keller M A, et al. Inhibition of triosephosphate isomerase by phosphoenolpyruvate in the feedback-regulation of glycolysis[J]. Open Biol, 2014, 4(3): 130232. doi: 10.1098/rsob.130232 [19] Tao G, Wen X, Wang X, et al. Bulk and single-cell transcriptome profiling reveal the metabolic heterogeneity in gastric cancer[J]. Sci Rep, 2023, 13(1): 8787. doi: 10.1038/s41598-023-35395-y [20] Mo C, Li H, Yan M, et al. Dopaminylation of endothelial TPI1 suppresses ferroptotic angiocrine signals to promote lung regeneration over fibrosis[J]. Cell Metab, 2024, 36(8): 1839-1857. e12. [21] Liu P, Sun S J, Ai Y J, et al. Elevated nuclear localization of glycolytic enzyme TPI1 promotes lung adenocarcinoma and enhances chemoresistance[J]. Cell Death Dis, 2022, 13(3): 205. doi: 10.1038/s41419-022-04655-6 -
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