Liver Research Group, Australian National University Medical School at The Canberra Hospital, Canberra, ACT, Australia.
J Gastroenterol Hepatol. 2013 Sep;28(9):1545-54. doi: 10.1111/jgh.12216.
By array-comparative genomic hybridization, we demonstrated cyclin E as one of seven genes associated with hepatocellular carcinoma (HCC) development in Ku70 DNA repair-deficient mice. We therefore explored the hypothesis that during hepatocarcinogenesis, cyclin E kinase can overcome the inhibitory effects of p53 and establish whether abnormal miRNA(mi-R)-34, a co-regulator of cyclin E and p53, can account for their interactions as "drivers" of HCC.
Dysplastic hepatocytes (DNs) and HCCs were generated from diethylnitrosamine (DEN)-injected C57BL/6 male mice at 3-12 months.
Cyclin E/cdk2 was barely expressed in normal liver, but was readily detected in dysplastic hepatocytes, localizing to glutathione-S transferase pi-form positive cells dissected by laser-dissection. Cyclin E kinase activity preceded cyclin D1, proliferating cell nuclear antigen expression in DNs and HCCs despite maximal p53 and p21 expression. We confirmed that cyclin E, rather than cyclin D1, is the proliferative driver in hepatocarcinogenesis by immunoprecipitation experiments demonstrating preferential binding of p21 to cyclin D1, allowing cyclin E-mediated "escape" from G1/S checkpoint. We then showed cyclin E was responsible for regulating wild-type p53 by knockdown experiments in primary HCC cells; cyclin E-knockdown increased p53 and p21, diminished anti-apoptotic Bcl-XL and reduced cell viability. Conversely, blocking p53 augmented cyclin E, Bcl-XL expression and increased proliferation. Physiological interactions between cyclin E/p53/p21 were confirmed in primary hepatocytes. miR-34a,c were upregulated in dysplastic murine, human liver and HCCs compared with normal liver, and appeared to be linked to cyclin E/p53.
Upregulation of functionally active cyclin E via miR34 with loss of p53 function is associated with cell-cycle checkpoint failure increasing proliferative drive that favors hepatocarcinogenesis.
通过阵列比较基因组杂交，我们发现细胞周期蛋白 E 是与 Ku70 DNA 修复缺陷型小鼠肝癌（HCC）发生相关的七个基因之一。因此，我们提出假说，即在肝癌发生过程中，细胞周期蛋白 E 激酶可以克服 p53 的抑制作用，并确定异常的 microRNA（miR）-34 是否可以作为细胞周期蛋白 E 和 p53 的共调控因子，将它们的相互作用解释为 HCC 的“驱动因素”。
采用二乙基亚硝胺（DEN）诱导 C57BL/6 雄性小鼠，在 3-12 个月时生成发育不良的肝细胞（DN）和 HCC。
细胞周期蛋白 E/cdk2 在正常肝脏中几乎不表达，但在发育不良的肝细胞中很容易检测到，其定位在通过激光切割分离的谷胱甘肽 S-转移酶 pi 阳性细胞中。尽管最大程度地表达了 p53 和 p21，但细胞周期蛋白 E 激酶活性先于细胞周期蛋白 D1 和增殖细胞核抗原在 DNs 和 HCC 中的表达。我们通过免疫沉淀实验证实，细胞周期蛋白 E 而不是细胞周期蛋白 D1 是肝癌发生过程中的增殖驱动因素，该实验表明 p21 优先与细胞周期蛋白 D1 结合，从而使细胞周期蛋白 E 介导的“逃逸”G1/S 检查点。然后，我们通过在原发性 HCC 细胞中进行敲低实验表明，细胞周期蛋白 E 负责调节野生型 p53；细胞周期蛋白 E 敲低增加了 p53 和 p21，减少了抗凋亡 Bcl-XL 并降低了细胞活力。相反，阻断 p53 增加了细胞周期蛋白 E、Bcl-XL 的表达并增加了增殖。在原代肝细胞中证实了细胞周期蛋白 E/p53/p21 之间的生理相互作用。与正常肝脏相比，发育不良的鼠、人类肝脏和 HCC 中上调了 miR-34a、c，并且似乎与细胞周期蛋白 E/p53 相关。
通过 miR34 上调具有功能活性的细胞周期蛋白 E，同时丧失 p53 功能，与细胞周期检查点失败相关，增加了有利于肝癌发生的增殖驱动力。
