| Objective: To investigate the effect and mechanism of saikosaponin d on autophagy of glioma C6 cells by miR-34 a.Method:1.The effect of saikosaponin d on the proliferation ability of glioma C6 cells was detected by CCK-8.2.According to the results of CCK-8 experiment,the glioma C6 cells were divided into Control(blank control)group and saikosaponin d(8 μmol/L)group.(1)The effect of saikosaponin d(8 μmol/L)on the proliferation ability of glioma C6 cells was detected by clone formation assay;(2)The effect of saikosaponin d(8 μmol/L)on the migration ability of glioma C6 cells was detected by scratches and transwell assay;(3)AO staining was used to detect the effect of saikosaponin d(8 μmol/L)on autophagy in glioma C6 cells;(4)Western blot was used to detect the protein expression of autophagy-related factors LC3-Ⅱ/LC3-Ⅰ,Beclin-1 and AKT/mTOR signaling pathway in glioma C6 cells in Control group and saikosaponin d(8 μmol/L)group;(5)The m RNA expression of autophagy-related factors LC3 and Beclin-1 and AKT/mTOR signaling pathways in glioma C6 cells in the Control group and saikosaponin d(8 μmol/L)group was detected by RT-qPCR.3.RT-qPCR was used to detect the expression of miR-34 a in glioma C6 cells in the Control group and the saikosaponin d(8 μmol/L)group.4.(1)The miR-34 a NC and miR-34 a inhibitor model was constructed in glioma C6 cells by transient transfection,and the transfection efficiency of fluorescently labeled(FAM)glioma C6 cells was observed under a fluorescence microscope;(2)RT-qPCR detection of miR-34 a expression in Control group,miR-34 a NC group,and miR-34 a inhibitor group;(3)Western blot was used to detect the protein expression of autophagy-related factors LC3-Ⅱ/LC3-Ⅰ and Beclin-1 and AKT/mTOR signaling pathways in glioma C6 cells in the control group,miR-34 a NC group and miR-34 a inhibitor group;(4)Western blot was used to detect the protein expression of autophagy-related factors LC3-Ⅱ/LC3-Ⅰ and Beclin-1 and AKT/mTOR signaling pathways in glioma C6 cells in the Control group,saikosaponin d(8μmol/L)group,saikosaponin d + miR-34 a NC group and saikosaponin d + miR-34 a inhibitor.Result:1.According to the results of CCK-8,compared with the Control(blank control)group,each concentration of saikosaponin d can inhibit the proliferation of glioma C6 cells,and the proliferation inhibition rate increases with the increase of drug concentration(P<0.01,P<0.001).2.(1)Compared with the Control group,after saikosaponin d(8 μmol/L)was applied to the glioma C6 cells for 24 h,the number of clones formed by the glioma C6 cells was significantly reduced(P<0.01);(2)According to scratches and Transwell experiments,compared with the Control group,saikosaponin d(8 μmol/L)group cell migration rate was significantly reduced(P<0.001);(3)According to the results of AO staining,saikosaponin d(8 μmol/L)group can significantly induce autophagy in glioma C6 cells;(4)According to the results of western blot,compared with the Control group,the protein expression levels of autophagy-related factors Beclin-1 and LC3-Ⅱ/LC3-Ⅰ in the saikosaponin d(8 μmol/L)group were significantly increased(both P<0.01),while the protein expression levels of p-AKT and p-mTOR decreased(both P<0.01),but had no significant effect on the expression level of AKT/mTOR total protein;(5)According to the results of RT-qPCR experiment,compared with the control group,the m RNA expression of autophagy-related factors Beclin-1 and LC3 in the saikosaponin d(8 μmol/L)group was significantly increased(P<0.01,P<0.001),while the m RNA expression of AKT and mTOR decreased significantly(P<0.05,P<0.001).3.According to the results of RT-qPCR experiments,compared with the Control group,the m RNA expression of miR-34 a in the saikosaponin d(8 μmol/L)treatment group was significantly increased(P<0.01).4.(1)The transfection rate of glioma C6 cells in the FAM-labeled miR-34 a NC group and the miR-34 a inhibitor group was observed under an inverted fluorescence microscope >80%;(2)According to the results of RT-qPCR experiment,Compared with the Control group and the miR-34 a NC group,the expression of miR-34 a in glioma C6 cells in miR-34 a inhibitor group was significantly decreased(both P<0.01);(3)Western blot results showed that compared with Control group and miR-34 a NC group,miR-34 a inhibitor It can reduce the protein expression levels of autophagy-related factors Beclin-1 and LC3-Ⅱ/LC3-Ⅰ in glioma C6 cells(P<0.05,P<0.01).At the same time,compared with Control and miR-34 a NC groups,miR-34 a inhibitor can significantly increase the protein expression levels of p-AKT and p-mTOR in glioma C6 cells(P<0.05,P<0.01);(4)As shown by the Western blot results,compared with the Control group,the expression levels of autophagy-related factors Beclin-1and LC3-Ⅱ/LC3-Ⅰ in the saikosaponin d(8 μmol/L)group and saikosaponin d + miR-34 a NC groups were significantly increased,and the protein expression levels of p-AKT and p-mTOR were significantly reduced(P<0.05,P<0.01),and compared with the saikosaponin d(8 μmol/L)group and saikosaponin d + miR-34 a NC groups,saikosaponin d + miR-34 a inhibitor groups can reduce the protein expression levels of autophagy-related factors Beclin-1 and LC3-Ⅱ/LC3-Ⅰ,and increase the protein expression levels of p-AKT and p-mTOR(P<0.05,P<0.01).Conclusion: After acting on glioma C6 cells,Bupleurum saponin d can increase miR-34 a to regulate AKT/mTOR signaling pathway,induce tumor cell autophagy,and inhibit their proliferation and migration. |
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