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目的:探讨羽扇豆醇对人膀胱癌T24细胞增殖的影响及对p53/miR-34a通路调控机制。方法:运用CCK-8法检测不同浓度羽扇豆醇(5~120μmol·L-1)分别作用24 h和48 h对T24细胞增殖的影响;运用CCK-8法结合Caspase抑制药确证参与羽扇豆醇诱导细胞死亡的Caspase亚型;分别运用荧光定量PCR(q PCR)和蛋白免疫印记评价羽扇豆醇对miR-34a及p53蛋白表达的影响;运用q PCR评价羽扇豆醇对miR-34a下游靶基因Bcl-2、CD44、c-Myc的mRNA表达的影响。结果:T24细胞经羽扇豆醇处理后,其细胞增殖受到明显抑制,且呈一定的剂量依赖性;药物作用24 h和48 h时羽扇豆醇的半抑制浓度(IC50)分别为(77.23±6.78),(64.58±4.23)μmol·L-1。与对照组相比,羽扇豆醇能够使T24细胞中p53蛋白表达上调,还能够增加miR-34a表达水平,差异具有统计学意义(P<0.01);羽扇豆醇处理后细胞内Bcl-2、CD44、c-Myc的mRNA表达量下调,差异具有统计学意义(P<0.01)。结论:羽扇豆醇具有抑制膀胱癌T24细胞增殖能力,其作用机制与调控p53/miR-34a通路有关。
Objective: To investigate the effects of lupulonol on the proliferation of human bladder cancer T24 cells and its mechanism of p53 / miR-34a pathway. Methods: The effects of different concentrations of lupulonol (5 ~ 120μmol·L-1) for 24 h and 48 h on proliferation of T24 cells were detected by CCK-8 assay. CCK-8 and Caspase inhibitors were used to determine the effects of lupulonol Caspase subtypes were selected to induce cell death. Fluorescent quantitative PCR (qPCR) and Western blotting were used to evaluate the expression of miR-34a and p53 protein. Luciferase assay was used to evaluate the effect of lupulonol on miR-34a downstream target genes Bcl-2, CD44, c-Myc mRNA expression. Results: Lupinol treatment inhibited the proliferation of T24 cells in a dose-dependent manner. The half-inhibitory concentrations (IC50) of lupulonol at 24 h and 48 h were (77.23 ± 6.78 ), (64.58 ± 4.23) μmol·L-1. Compared with the control group, lupeol could upregulate the expression of p53 protein in T24 cells and increase the expression level of miR-34a (P <0.01). After lupine treatment, the levels of Bcl-2, CD44, c-Myc mRNA was down-regulated, the difference was statistically significant (P <0.01). Conclusion: Lupine alcohol can inhibit the proliferation of bladder cancer T24 cells and its mechanism is related to the regulation of p53 / miR-34a pathway.