基于CKB/p53信号通路探讨白花丹醌对人肝癌细胞增殖和凋亡的影响

Effects of plumbagin on proliferation and apoptosis of human hepatocellular carcinoma cells based on CKB/p53 signaling pathway

基于CKB/p53信号通路研究白花丹醌对人肝癌Huh-7细胞增殖和凋亡的影响及其作用机制。将白花丹醌1~12μmol·L^(-1)处理Huh-7细胞,进行细胞增殖和活性cell counting kit-8(CCK-8)实验,确定1、3、6μmol·L^(-1)为低、中、高浓度用于后续实验。采用规律成簇的间隔短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)/CRISPR相关蛋白(CRISPR-associated proteins,Cas)-9基因编辑技术敲除Huh-7细胞中的脑型肌酸激酶(creatine kinase,brain type,CKB)基因;CKB过表达慢病毒转染Huh-7细胞上调CKB的表达。通过平板克隆实验检测细胞增殖;流式细胞术检测细胞凋亡;实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)检测CKB的mRNA表达情况;蛋白免疫印迹(Western blot,WB)实验检测CKB、p53、双微体同源基因2(mouse double minute 2 homolog,MDM2)、B淋巴瘤细胞瘤2(B-cell lymphoma 2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)和半胱天冬酶(caspase)-3蛋白表达水平。结果显示白花丹醌显著抑制Huh-7细胞增殖,诱导细胞凋亡。与对照组比较,白花丹醌组凋亡水平显著升高,而白花丹醌联合凋亡抑制剂组凋亡水平显著降低。白花丹醌可显著下调CKB、Bcl-2和MDM2蛋白表达水平,上调p53、Bax和caspase-3蛋白表达水平。敲除CKB基因后,Huh-7细胞增殖能力降低,凋亡率升高,Bcl-2和MDM2蛋白表达水平降低,p53、Bax和caspase-3蛋白表达水平升高。上调CKB表达后,Huh-7细胞增殖能力增强,Bcl-2和MDM2蛋白表达水平升高,p53、Bax和caspase-3蛋白表达水平降低。而白花丹醌可逆转过表达CKB对Huh-7细胞增殖和凋亡的作用。该实验表明,白花丹醌可显著抑制Huh-7细胞的增殖能力,其机制可能是通过抑制CKB表达,激活p53信号通路,调控线粒体相关凋亡蛋白的表达,最终诱导细胞凋亡。

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 12μmol·L^(-1) for cell counting kit-8(CCK-8)assay,and 1,3,and 6μmol·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.