SYB Inducethe P53/caspase9 Axis To Regulate Apoptosis In HepG2 Liver Cancer Cells

Section 1 Effect of SYB on liver cancer growth.Objective:The effect of safflower yellow B on growth and proliferation of hepatocellular carcinoma cells was studied.Methods:The growth percentage effect by SYB(50,100,150,200nmol/ml)at12h,24h,36h and 48h was detected by CCK-8.The apoptosis rate was detected by Annexin V/PI.Results:With the increase of SYB time,the growth rate of HepG2 cells gradually changed.The growth of HepG2 cells was the worst at 150nmol/ml at 36h.There was a significant difference between the SYB 0 groups and the other groups on the apoptosis rate of HepG2.In the late apoptosis rate,150 nmol/ml,and 200 nmol/ml were significantly different from that of the control group,which was dose dependent.Conclusions:SYB can inhibit the growth of HepG2 cells,inhibit the proliferation of HepG2 cells and induce the apoptosis of tumor cells.Section 2 The miR34a expression after SYB effect on the HepG2.Objective:SYB in the miRNA after the change of the more intense miRNA and its expression analysis.Methods:The expression of miRNA was detected by gene chip after 150nmol/m1SYB acting on hepatoma cell line HepG2.Quantitative PCR was used to study the expression of miRNA.Results:There are 19 miRNA affected by SYB.The most obvious change was miR-34a.The expression levels of 20min,40min and miR-34a in 60min were significantly higher than those of Omin expression in mRNA(P<0.05).The expression level of 40min and 60min in mRNA level was significantly higher than that of 20 min in miR-34a(P<0.05).There was no significant difference in the expression level of 40min and 60min at mRNA level in miR-34a(P<0.05).Conclusions:After SYB was used with 40min,the expression level of miR-34a at the mRNA level was the best.Section 3 The p53,caspase3(cleaved),caspase 9,caspase 8,Bcl-2 expression after SYB effect on the HepG2.Objective:A preliminary study on the expression of p53 and mitochondrial pathway in HepG2 cells after SYB.Methods:The p53,caspase3(cleaved),caspase 9,caspase 8,Bcl-2 expression were detected by western blot.Results:With the effect time increase of SYB,the expression of Bcl-2 decreased gradually.The expression of 20min in Bcl-2 was not significantly different from that of Omin.The Bcl-2 expression of Omin and 60min was significantly lower than Omin.There was no significant difference in Bcl-2 expression between 40min and 60min.The p53,caspase3(cleaved),caspase 9,caspase 8,Bcl-2 expression were increasing.The expression levels of 20min,40min and 60min were significantly higher than Omin.There was no significant difference between 40min and 60min.Conclusions:SYB can induce HepG2 apoptosis,possibly through the following pathways:SYB enhanced the transcription level of p53,activating transcription of Bax downstream,inhibit the transcription of Bcl-2,the expression of p53 protein and Bax protein,and Bcl-2 protein expression decreased,may be the p53 protein and Bcl-2 protein combined,or Bcl-2 itself has formed two dimers,the mitochondrial membrane potential or specific pore opening,the release of cytochrome C,further caused the caspase cascade,resulting in cell apoptosis.Section 4 miR34a and caspase9 expression after p53 inhibited.Objective:p53 the role of miR-34a in this process,and the effects of other ideas on protein expression.Methods:After 150 nM of p53 inhibitor was used in the HepG2 of hepatoma cell line 12h,150nmol/1 was treated with SYB at the final concentration of HepG2,and the cells were collected with 0,20min,40min and 60min to detect miRNA.Do Western experiment.The proliferation of SYB group,SYB plus p53 inhibitor group,and no SYB group were detected.Results:After the addition of p53 inhibitor,the expression level of miR34a at the mRNA level was almost no change in each time point.The p53,caspase3(cleaved),caspase 9,caspase 8,Bcl-2 expression were no change.With the time increasing,the proliferation rate of HepG2 cells also increased in 24,36 and 48h cell percentage was significantly higher than that of 12h(P<0.05),cell proliferation and 48h percentage of 36 was significantly higher than that of 24h(P<0.05),but there was no obvious change in the percentage of cell proliferation and 48H 36(P>0.05).Conclusions:p53 can regulate the expression of miR-34a.Section 5 p53 and caspase9 expression after miR34a inhibited.Objective:The changes of p53 and caspase9 and their related proteins were studied after miR34a inhibitied.Methods:RNA interference was used to inhibit miR34a.The expression of p53,Caspase3(cleaved),caspase 9,caspase 8,Bcl-2 was detected.CCK-8 was used to detect the proliferation of HepG2.Results:When miR34a was inhibited,the bands of Bcl-2 did not gradually become shallower with the increase of SYB time,and the expression of 20min in Bcl-2 was not significantly different from Omin.The Bcl-2 expression of 40min and 60min was significantly lower than Omin.The p53,caspase3(cleaved),caspase 9,caspase 8,Bcl-2 expression were increasing.The expression of caspase 8 in 40min and 60min was significantly higher than in Omin.The proliferation rate of HepG2 was also increased.And the percentage of cell proliferation in 24,36 and 48h was significantly higher than that of 12h.There was no significant change in cell proliferation percentage of 36h and 48h.Conclusions:MiR-34a regulates apoptosis through regulation of Bcl-2.