METHODS: We constructed a eukaryotic expression vector and purified the recombinant protein BCT. CCK8 used to analyzed the activity of EC after treated by different concentration of BCT. UPLC-MS/MS and ELISA used to detect the metabonomics and metabolites. The ability of migration and invasion was completed by transwell assay. RT-QPCR, WB used to analyze the expression of relevant genes.
RESULTS: Our data showed that BCT was higher expression in EC tumor tissues (p < 0.05) and BCT in 20 µg/mL promoted the survival, invasion and migration of EC cells (EC109) (p < 0.05). Meanwhile, UPLC-MS/MS results suggested that BCT resulted in an augmentation of hypotaurine metabolism, arachidonic acid metabolism, glycolysis/gluconeogenesis, tryptophan metabolism, citrate cycle activity in EC109. The metabolic changes resulted in decreasing in glucose and pyruvate levels but increase in lactate dehydrogenase (LDH) activity and lactic acid (LA) as well as the expression of glucose transporter 1, Hexokinase 2, LDH which regulated the glycolysis were all changed (p < 0.05). The BCT treatment upregulated the expression of TLR4, Akt, HIF-1α (p < 0.05) which regulated the production of LA. Furthermore, LA stimulation promoted the expression of GPR81, Wnt, and β-catenin (p < 0.05), thereby inducing EMT and metastasis in EC109 cells.
CONCLUSIONS: Altogether, these findings identified that impact of BCT in regulation of glycolysis in EC109 and its involves the TLR4/Akt/HIF-1α pathway. Meanwhile, glycolysis increasing the release of LA and promote the EMT of EC109 by GPR81/Wnt/β-catenin signaling pathway. In summary, our findings underscore the potential of targeting BCT as an innovative strategy to mitigate the development of EC.
方法:构建真核表达载体,纯化重组蛋白BCT。CCK8用于分析不同浓度BCT处理后EC的活性。UPLC-MS/MS和ELISA检测代谢组学和代谢产物。通过transwell测定完成迁移和侵袭能力。RT-QPCR,WB用于分析相关基因的表达。
结果:我们的数据表明,BCT在EC肿瘤组织中的表达更高(p<0.05),BCT在20µg/mL中促进了生存率,EC细胞的侵袭和迁移(EC109)(p<0.05)。同时,UPLC-MS/MS结果表明,BCT导致了低牛磺酸代谢的增强,花生四烯酸代谢,糖酵解/糖异生,色氨酸代谢,EC109中的柠檬酸循环活性。代谢变化导致葡萄糖和丙酮酸水平降低,但乳酸脱氢酶(LDH)活性和乳酸(LA)活性增加以及调节糖酵解的葡萄糖转运蛋白1,己糖激酶2,LDH的表达均发生变化(p<0.05)。BCT处理上调TLR4、Akt、HIF-1α(p<0.05)调节LA的产生。此外,LA刺激促进GPR81、Wnt、和β-连环蛋白(p<0.05),从而在EC109细胞中诱导EMT和转移。
结论:总而言之,这些发现确定了BCT在调节EC109糖酵解中的影响,其涉及TLR4/Akt/HIF-1α途径。同时,糖酵解通过GPR81/Wnt/β-catenin信号通路增加LA的释放并促进EC109的EMT。总之,我们的研究结果强调了靶向BCT作为缓解EC发展的创新策略的潜力.