Magnesium isoglycyrrhizinate suppresses bladder cancer progression by modulating the miR-26b/Nox4 axis

Magnesium isoglycyrrhizinate (MI), a magnesium salt of 18α-GA stereoisomer, has been reported to exert efficient hepatoprotective activity. However, its effect on bladder cancer remains unclear. The study explored the effects of MI on the growth, colony formation, apoptosis, invasion, and migration of bladder cancer cells (HTB9 and BIU87 cells). Typical apoptotic changes of bladder cancer cells such as nuclear concentration and fragmentation were observed using Hoechst staining. The effects of MI on the expression levels of microRNA-26b (miR-26b), NADPH oxidase 4 (Nox4), nuclear transcription factor-κB (NF-κB), and hHypoxia inducible factor-1α (HIF-1α) were detected using qRT-PCR and Western blot. The potential targets of miR-26b were predicted using Targetscan, and their interactions were determined by luciferase reporter assay. A xenograft mouse model was established to evaluate the anti-tumor effects of MI <i>in vivo</i>. MI significantly suppressed the proliferation, colony formation, invasion, and migration and induced apoptosis of human bladder cancer cells, and MI significantly increased miR-26b expression. Nox 4 was identified to be a direct target of miR-26b. MiR-26b mimics significantly decreased the relative luciferase activity of wild type (WT) Nox 4 but not mutant type (MUT) Nox4. Meanwhile, MI markedly downregulated the expression levels of Nox4, NF-κB, and HIF-1α both <i>in vitro</i> and <i>in vivo</i>. Moreover, MI inhibited xenograft tumor growth <i>in vivo</i> and decreased the expression of Nox4, NF-κB, and HIF-1α. Overall, MI showed a potent anti-tumor effect against bladder cancer partially via modulating the miR-26b/Nox4 axis.

异甘草酸镁（Magnesium isoglycyrrhizinate，MI）作为18α-甘草酸立体异构体的镁盐，已被报道具有高效的保肝活性。然而其对膀胱癌的作用仍不明确。本研究探讨了MI对膀胱癌细胞（HTB9与BIU87细胞）的增殖、集落形成、凋亡、侵袭及迁移能力的影响。通过Hoechst染色（Hoechst staining）观察到膀胱癌细胞出现核固缩、碎裂等典型凋亡形态学改变。采用qRT-PCR与Western blot检测了MI对microRNA-26b（miR-26b）、烟酰胺腺嘌呤二核苷酸磷酸氧化酶4（NADPH oxidase 4，Nox4）、核转录因子-κB（NF-κB）以及缺氧诱导因子-1α（hypoxia inducible factor-1α，HIF-1α）表达水平的影响。利用Targetscan预测miR-26b的潜在靶基因，并通过荧光素酶报告基因实验（luciferase reporter assay）验证二者的相互作用。构建异种移植小鼠模型以体内评价MI的抗肿瘤作用。结果显示，MI可显著抑制人膀胱癌细胞的增殖、集落形成、侵袭与迁移，并诱导细胞凋亡，同时显著上调miR-26b的表达。Nox4被鉴定为miR-26b的直接靶基因。miR-26b模拟物可显著降低野生型（WT）Nox4的相对荧光素酶活性，但对突变型（MUT）Nox4无此作用。与此同时，MI在体外（in vitro）与体内（in vivo）均能显著下调Nox4、NF-κB及HIF-1α的表达水平。此外，MI可抑制异种移植瘤的体内生长，并降低瘤组织中Nox4、NF-κB及HIF-1α的表达。综上，MI对膀胱癌具有显著的抗肿瘤作用，其机制部分通过调控miR-26b/Nox4信号轴实现。