尿素循环限速酶CPS1对肝癌细胞增殖和凋亡的影响及机制研究

Mechanism and effect of urea cycle rate-limiting enzyme CPS1 upon hepatocellular carcinoma cell proliferation and apoptosis

目的探讨尿素循环限速酶CPS1影响肝癌细胞增殖及凋亡的可能机制。方法用慢病毒分别构建CPS1高表达(Hepa1-6-CPS1)和低表达(Huh7-shCPS1)的稳转肝癌细胞系;细胞生长实验和克隆形成实验评价CPS1对肝癌细胞增殖能力的影响;流式细胞术检测细胞凋亡;小鼠皮下移植瘤模型验证CPS1对肝癌生长的影响,免疫组织化学法检测皮下瘤组织中Ki-67及CPS1含量;Western blotting检测肝癌细胞中CPS1及c-Myc的表达量。结果与正常肝细胞相比,CPS1在肝癌细胞中的表达均有所下调(P<0.05);与阴性对照组比较,CPS1在Hepa1-6-CPS1和Huh7-shCPS1细胞中的表达量分别显著上调和下调(P均<0.05);细胞生长实验及克隆形成实验结果显示,CPS1低表达或高表达分别促进或抑制肝癌细胞生长(P<0.05),增加或减少其克隆形成数(P<0.001);流式细胞术检测结果显示,CPS1低表达的肝癌细胞凋亡能力被抑制(P<0.001),而CPS1高表达则促进肝癌细胞凋亡(P<0.05);与阴性对照组比较,CPS1高表达的皮下移植瘤生长减慢,皮下瘤组织中CPS1表达量与Ki-67呈负相关(P<0.05);Western blotting检测结果显示,CPS1低表达促进c-Myc表达上调(P<0.001),而CPS1高表达则抑制c-Myc表达(P<0.001)。结论尿素循环限速酶CPS1通过调控c-Myc信号通路抑制肝癌细胞增殖,促进肝癌细胞凋亡。

Objective To explore the possible mechanism of the urea cycle rate-limiting enzyme CPS1 affecting the proliferation and apoptosis of liver cancer cells.Methods Western blotting was used to detect the expression of CPS1 in different liver cancer cells,and CPS1 overexpression(Hepa1-6-CPS1)and knockdown(Huh7-shCPS1)stable liver cancer cell lines were constructed with lentivirus.Cell growth experiment and clonies formation experiment was used to evaluate the effect of CPS1 on the proliferation of liver cancer cells.Cell apoptosis was detected by flow cytometry.The effect of CPS1 on the growth of liver cancer in vivo was verified by subcutaneous transplanted tumor model in mice.The immunohistochemical method was used to detect the cotent of Ki-67 and CPS1 in subcutaneous tumor tissue.Western blotting was used to detect the changes of c-Myc and CPS1 protein level.Results Compared with the normal hepatocytes,the expression of CPS1 in liver cancer cells was universally down-regulated(P<0.05).Compared with the negative control group,the protein of CPS1 in Hepa1-6-CPS1 and in Huh7-shCPS1 were respectively up-regulated and down-regulated(P<0.05).The results of cell growth experiments and clone formation experiments suggested that low or high expression of CPS1 respectively promoted or inhibited the growth of liver cancer cells(P<0.05),and increased or decreased the numbers of clone formation respectively(P<0.001).The flow cytometry results indicated that the apoptotic ability of liver cancer cells with low expression of CPS1 was inhibited(P<0.001),while high expression of CPS1 promoted the apoptosis of liver cancer cells(P<0.05).Compared with the negative control group,the growth of subcutaneous transplanted tumors with high expression of CPS1 slowed down,and the expression of CPS1 in subcutaneous tumor tissue was negatively correlated with Ki-67(P<0.05).Western blotting results suggested that low expression of CPS1 promoted the up-regulation of c-Myc(P<0.001),while the high expression of CPS1 inhibited the expression of c-Myc(P<0.001).Conclusion Urea cycle rate-limiting enzyme CPS1 inhibits the proliferation of hepatoma cells and promotes the apoptosis of hepatoma cells by regulating c-Myc signaling pathway.