五味子甲素调控miR-873-5p/G6PD轴对胃癌SGC-7901细胞活力、凋亡及有氧糖酵解的影响

The effect of Schisandrin A on miR-873-5p/G6PD axis regulation on the viability,apoptosis,and aerobic glycolysis of gastric cancer SGC-7901 cells

目的:探究五味子甲素调控微小RNA-873-5p(miR-873-5p)/葡萄糖-6-磷酸脱氢酶(G6PD)轴对胃癌SGC-7901细胞活力、凋亡及有氧糖酵解的影响。方法:将胃癌SGC-7901细胞分成SGC-7901组、低浓度五味子甲素组、高浓度五味子甲素组、高浓度五味子甲素+空载质粒组、高浓度五味子甲素+miR-873-5p沉默组。各组给予相应药物干预及转染,培养48 h。细胞计数试剂盒-8和流式细胞术分别检测细胞活力和凋亡率;试剂盒检测葡萄糖消耗、乳酸产生以及三磷酸腺苷(ATP)生成;实时荧光定量PCR法检测miR-873-5p和G6PD mRNA表达;蛋白印迹法检测G6PD及凋亡相关蛋白表达;双荧光素酶报告基因检测miR-873-5p与G6PD的靶向关系。结果:与SGC-7901组比较,低、高浓度五味子甲素组细胞活力、B淋巴细胞瘤-2(Bcl-2)蛋白、葡萄糖消耗、乳酸产生、ATP生成相对水平、G6PD mRNA和蛋白水平显著降低,细胞凋亡率、Bcl-2相关X蛋白(Bax)、活化的半胱氨酸天冬氨酸蛋白酶-3(cleaved-Caspase-3)蛋白、miR-873-5p水平显著升高(P<0.05);高浓度五味子甲素组细胞活力、Bcl-2蛋白、葡萄糖消耗、乳酸产生、ATP生成相对水平、G6PD mRNA和蛋白水平显著低于低浓度五味子甲素组,细胞凋亡率、Bax、cleaved-Caspase-3蛋白、miR-873-5p水平显著高于低浓度五味子甲素组(P<0.05);与高浓度五味子甲素组和高浓度五味子甲素+空载质粒组比较,高浓度五味子甲素+miR-873-5p沉默组细胞活力、Bcl-2蛋白、葡萄糖消耗、乳酸产生、ATP生成相对水平、G6PD mRNA和蛋白水平显著升高,细胞凋亡率、Bax、cleaved-Caspase-3蛋白、miR-873-5p水平显著降低(P<0.05);miR-873-5p可靶向调控G6PD的表达。结论:五味子甲素可通过上调miR-873-5p来靶向下调G6PD表达,从而抑制胃癌SGC-7901细胞活力和有氧糖酵解,促进其凋亡。

Objective To investigate the effect of Schisandrin A on the regulation of microRNA-873-5p(miR-873-5p)/glucose-6-phosphate dehydrogenase(G6PD)axis on the viability,apoptosis,and aerobic glycolysis of gastric cancer SGC-7901 cells.Methods Divide gastric cancer SGC-7901 cells into SGC-7901 group,low concentration Schisandrin A group,high concentration Schisandrin A group,high concentration Schisandrin A+empty plasmid group,and high concentration Schisandrin A+miR-873-5p silencing group.Each group was given corresponding drug intervention and transfection,and cultured for 48 hours.Cell viability and apoptosis rate were detected using cell count kit-8 and flow cytometry,respectively;the reagent kit detects glucose consumption,lactate production,and adenosine triphosphate(ATP)production;real-time fluorescence quantitative PCR was used to detect the expression of miR-873-5p and G6PD mRNA;western blot was used to detect the expression of G6PD and apoptosis-related proteins;targeting relationship between miR-873-5p and G6PD detected by dual luciferase reporter gene detection.Results Compared with SGC-7901 group,the cell viability,B cell lymphoma-2(Bcl-2)protein,glucose consumption,lactate production,ATP production relative levels,G6PD mRNA and protein levels in low and high concentration Schisandrin A groups were significantly decreased,apoptosis rate,Bcl-2 related X protein(Bax),cleaved-Caspase-3 protein and miR-873-5p levels were significantly increased(P<0.05);The cell viability,Bcl-2 protein,glucose consumption,lactate production,ATP production relative levels,G6PD mRNA and protein levels in the high concentration Schisandrin A group were significantly lower than those in the low concentration Schisandrin A group,the cell apoptosis rate,Bax,cleaved-Caspase-3 protein,miR-873-5p levels were significantly higher than those in the low concentration Schisandrin A group(P<0.05);Compared with high concentration Schisandrin A group and high concentration Schisandrin A+empty plasmid group,the cell viability,Bcl-2 protein,glucose consumption,lactate production,ATP production relative levels,G6PD mRNA and protein levels in the high concentration Schisandrin A+miR-873-5p silencing group were significantly increased,the cell apoptosis rate,Bax,cleaved-Caspase-3 protein,miR-873-5p levels were significantly decreased(P<0.05);miR-873-5p can target and regulate the expression of G6PD.Conclusion Schisandrin A can target and downregulate G6PD expression by upregulating miR-873-5p,thereby inhibiting the viability and aerobic glycolysis of gastric cancer SGC-7901 cells,and promoting their apoptosis.