大豆苷元影响非小细胞肺癌细胞增殖和凋亡:p53信号通路的作用

Daidzein affects proliferation and apoptosis in non-small cell lung cancer cells:role of p53 signaling pathway

目的研究大豆苷元(daidzein,DD)对非小细胞肺癌细胞增殖和凋亡的影响,并重点探讨p53信号通路在其中可能发挥的作用。方法CCK-8法和流式细胞术检测大豆异黄酮和DD对HELF和H1299细胞活力和凋亡的影响;基因芯片技术检测DD处理H1299细胞后基因的表达量变化,GSEA和差异分析筛选主要通路和关键基因;RT-qPCR和Western blot实验验证主要通路中关键基因(p53和CASP9)的mRNA和蛋白表达差异;p53抑制剂Pifithrin-α抑制p53的表达后,检测DD对p53 mRNA和蛋白表达水平的影响,并观察其对H1299细胞的增殖效应。结果大豆异黄酮和DD能促进肺正常细胞增殖和抑制其凋亡,且能抑制肺癌细胞增殖和促进其凋亡。p53信号通路在DD处理组中明显富集(NES=1.78,P=0.000),且发现处理组中p53和CASP9基因的表达出现明显上调。与对照组相比,DD处理的HELF和H1299细胞中CASP9和p53的mRNA表达均明显提高(P<0.05),HELF细胞中p53蛋白表达亦出现增加(P<0.05)。抑制p53表达后,DD可明显增加H1299和HELF细胞中p53的mRNA表达(P<0.05),亦可明显提高H1299细胞中p53蛋白的表达(P<0.05),并观察到DD可抑制肺癌细胞的增殖。结论DD能抑制肺癌H1299细胞的增殖并促进其凋亡,且其机制主要涉及p53信号通路。

Aim To investigate the effects of daidzein(DD)on the proliferation and apoptosis of non-small cell lung cancer cells,with a focus on the possible role of the p53 signaling pathway in this regard.Methods CCK-8 method and flow cytometry were used to detect the effects of soy isoflavone crude extract and DD on the viability and apoptosis of HELF and H1299 cells.Gene microarray was used to detect the changes in gene expression after treatment of H1299 cells with DD.GSEA and differential analysis were used to screen the major pathways and key genes.RT-qPCR and Western blot were performed to verify the differences in mRNA and protein expression of key genes(p53 and CASP9)in the major pathways.After p53 inhibitor Pifithrin-αinhibited the expression of p53,the effect of DD on p53 mRNA and protein expression levels was examined,and the proliferative effect on H1299 cells was observed.Results Soy isoflavone crude extract and DD promoted proliferation and inhibited apoptosis of normal lung cells and inhibited proliferation and promoted apoptosis of lung cancer cells.p53 signaling pathway was significantly enriched in the DD-treated group(NES=1.78,P=0.000),and the expressions of p53 and CASP9 genes were found to be significantly up-regulated in the treated group.Compared with the control group,mRNA expression of CASP9 and p53 significantly increased in both HELF and H1299 cells treated with DD(P<0.05),and p53 protein expression also increased in HELF cells(P<0.05).After inhibition of p53 expression,DD significantly increased the mRNA expression of p53 in H1299 and HELF cells(P<0.05)and also markedly increased the expression of p53 protein in H1299 cells(P<0.05),and it was observed that DD inhibited the proliferation of lung cancer cells.Conclusions DD inhibits the proliferation and promotes the apoptosis of lung cancer H1299 cells,and the mechanism mainly involves the p53 signaling pathway.