Effects of plumbagin on proliferation and apoptosis of human hepatocellular carcinoma cells based on CKB/p53 signaling pathway
To investigate the effects of plumbagin on the proliferation and apoptosis of human hepatoma Huh-7 cells and its mechanism based on the creatine kinase B(CKB)/p53 signaling pathway.Huh-7 cells were treated with plumbagin from 1 to 12 μmol·L-1 for cell counting kit-8(CCK-8)assay,and 1,3,and 6 μmol·L-1 were determined as low,medium,and high concentrations of plumbagin for subsequent experiments.CKB gene was knocked out in Huh-7 cells by clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated proteins(Cas)-9 gene editing technology.CKB overexpression lentivirus was transfected into Huh-7 cells to up-regulate the expression of CKB.Cell proliferation and apoptosis were detected by plate cloning assay and flow cytometry.The mRNA expression of CKB was detected by quantitative real-time PCR(qRT-PCR).CKB,p53,mouse double minute 2 homolog(MDM2),B-cell lymphoma 2(Bcl-2),Bcl-2 associated X protein(Bax),and caspase-3 protein were detected by Western blot(WB).The results showed that plumbagin significantly inhibited the proliferation of Huh-7 cells and induced cell apoptosis.Compared with the control group,the apoptosis level was significantly increased in the plumbagin group,while the apoptosis level was significantly decreased in the plumbagin combined with the apoptosis inhibitor group.Plumbagin significantly down-regulated the protein expression levels of CKB,Bcl-2,and MDM2 and up-regulated the protein expression levels of p53,Bax,and caspase-3.Knockdown of the CKB gene decreased the proliferative ability of Huh-7 cells,increased the apoptotic rate,decreased the expression levels of Bcl-2 and MDM2 proteins,and increased the expression levels of p53,Bax,and caspase-3 proteins.After up-regulation of CKB expression,the proliferation ability of Huh-7 cells was enhanced,and the protein expression levels of Bcl-2 and MDM2 were elevated.The protein expression levels of p53,Bax,and caspase-3 were decreased.In addition,plumbagin reversed the effect of overexpression of CKB on the proliferation and apoptosis of Huh-7 cells.In conclusion,plumbagin significantly inhibited the proliferative ability of Huh-7 cells,and the mechanism may be related to the inhibition of CKB expression,activation of the p53 signaling pathway,and regulation of the expression of mitochondrial-associated apoptotic proteins,ultimately inducing cell apoptosis.
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