醉茄素A通过circOSBPL10/miR-128-3p通路调控乳腺癌MDA-MB-231细胞糖酵解、增殖、迁移及侵袭的机制研究

Molecular mechanism of withaferin A regulating glycolysis, proliferation, migration and invasion of breast cancer MDA-MB-231 cells through the circOSBPL10/miR-128-3p pathway

关键词:  醉茄素A  circOSBPL10  miR-128-3p  乳腺癌  糖酵解

Key words:  withaferin A  circOSBPL10  miR-128-3p  breast cancer  glycolysis

摘要:

目的 探讨醉茄素A对乳腺癌MDA-MB-231细胞糖酵解、增殖、迁移、侵袭的影响及其对环状RNAOSBPL10（circOSBPL10）/微小RNA-128-3p（miR-128-3p）通路的调控作用。方法 采用不同浓度（10、20、40 μg/mL）的醉茄素A处理乳腺癌细胞MDA-MB-231（醉茄素A低剂量组、醉茄素A中剂量组、醉茄素A高剂量组）；si-NC、si-circOSBPL10分别转染入MDA-MB-231细胞（si-NC组、si-circOSBPL10组）；pcDNA、pcDNA-circOSBPL10分别转染入MDA-MB-231细胞后加入40 μg/mL的醉茄素A处理细胞（醉茄素A+pcDNA组、醉茄素A+pcDNA-circOSBPL10组）；根据试剂盒检测葡萄糖消耗与乳酸产生量，以及丙酮酸激酶、己糖激酶的水平；采用噻唑蓝（MTT）法检测细胞增殖抑制率；采用Transwell小室实验检测细胞迁移及侵袭能力；采用实时荧光定量聚合酶链反应（qRT-PCR）法检测circOSBPL10与miR-128-3p的表达量；双荧光素酶报告基因实验检测circOSBPL10与miR-128-3p的靶向关系；Western blot法检测增殖标记蛋白细胞增殖核抗原-67（Ki-67）、基质金属蛋白酶-2（MMP-2）、基质金属蛋白酶-9（MMP-9）蛋白表达量。结果 与对照组比较，醉茄素A低剂量组、醉茄素A中剂量组、醉茄素A高剂量组葡萄糖消耗与乳酸生成量以及丙酮酸激酶、己糖激酶的水平均明显降低（P＜0.05），迁移及侵袭细胞数减少（P＜0.05），细胞增殖抑制率升高（P＜0.05），Ki-67、MMP-2、MMP-9蛋白水平降低（P＜0.05），circOSBPL10的表达水平降低（P＜0.05），miR-128-3p的表达水平升高（P＜0.05）；双荧光素酶报告基因实验证实circOSBPL10可靶向结合miR-128-3p；与si-NC组比较，si-circOSBPL10组葡萄糖消耗与乳酸生成量以及丙酮酸激酶、己糖激酶的水平均明显降低（P＜0.05），细胞增殖抑制率升高（P＜0.05），迁移及侵袭细胞数减少（P＜0.05），Ki-67、MMP-2、MMP-9蛋白水平降低（P＜0.05）；与醉茄素A+pcDNA组比较，醉茄素A+pcDNA-circOSBPL10组葡萄糖消耗与乳酸生成量以及丙酮酸激酶、己糖激酶的水平均明显升高（P＜0.05），细胞增殖抑制率降低（P＜0.05），迁移及侵袭细胞数增多（P＜0.05），Ki-67、MMP-2、MMP-9蛋白水平升高（P＜0.05）。结论 醉茄素A可通过调控circOSBPL10/miR-128-3p通路而抑制乳腺癌细胞糖酵解、增殖、迁移及侵袭。

Abstract:

Objective To explore the effect of withaferin A on glycolysis, proliferation, migration and invasion of breast cancer MDA-MB-231 cells and its regulation of circOSBPL10/miR-128-3p pathway.Methods Different concentrations (10, 20, 40 μg/mL) of withaferin A were used to treat breast cancer cells MDA-MB-231 (withaferin A low dose group, withaferin A medium dose group, withaferin A high dose group). si-NC and si-circOSBPL10 were transfected into MDA-MB-231 cells (si-NC group, si-circOSBPL10 group). After pcDNA and pcDNA-circOSBPL10 were transfected into MDA-MB-231 cells respectively, 40 μg/mL withaferin A was added to the cells (bubnerin A+pcDNA group, bubnerin A+pcDNA-circOSBPL10 group). Glucose consumption, lactic acid production and the levels of pyruvate kinase and hexokinase were detected according to the kit. The MTT method was used to detect the inhibition rate of cell proliferation. Transwell chamber experiment was used to detect cell migration and invasion ability. The qRT-PCR method was used to detect the expression of circOSBPL10 and miR-128-3p. The dual luciferase reporter gene experiment was used to detect the targeting relationship between circOSBPL10 and miR-128-3p. Western blot method was used to detect the protein expression of Ki-67, MMP-2 and MMP-9.Results Compared with the control group, glucose consumption, lactate production and the levels of pyruvate kinase and hexokinase in the withaferin A low-dose group, withaferin A medium-dose group, and withaferin A high-dose group were significantly reduced (P < 0.05), and the number of migrating and invasive cells was decreased (P < 0.05), and the protein levels of Ki-67, MMP-2, MMP-9 were decreased (P < 0.05), and the expression level of circOSBPL10 was decreased (P < 0.05), while the cell proliferation inhibition rate was increased (P < 0.05), and the expression level of miR-128-3p was increased (P < 0.05). The dual luciferase reporter gene experiment confirmed that circOSBPL10 could target miR-128-3p. Compared with the si-NC group, glucose consumption, lactate production and the levels of pyruvate kinase and hexokinase in the si-circOSBPL10 group were significantly reduced(P < 0.05), and cell proliferation inhibition rate was increased (P < 0.05), and the number of migration and invasion cells was decreased (P < 0.05), and the protein levels of Ki-67, MMP-2, and MMP-9 were decreased (P < 0.05). Compared with the withaferin A+pcDNA group, the glucose consumption, lactate production and the levels of pyruvate kinase and hexokinase in the withaferin A+pcDNA-circOSBPL10 group were significantly increased (P < 0.05), and the number of migration and invasion cells was increased (P < 0.05), and the protein levels of Ki-67, MMP-2 and MMP-9 were increased (P < 0.05), while the cell proliferation inhibition rate was reduced (P < 0.05).Conclusion withaferin A could inhibit glycolysis, proliferation, migration and invasion of breast cancer cells by regulating the circOSBPL10/miR-128-3p pathway.