Cyclin E facilitates dysplastic hepatocytes to bypass G1/S checkpoint in hepatocarcinogenesis
Cyclin E facilitates dysplastic hepatocytes to bypass G1/S checkpoint in hepatocarcinogenesis
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DOI：
10.1111/jgh.12216
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发表时间：
2013-09-01
影响因子：
4.1
通讯作者：
Teoh, Narci C.
中科院分区：
文献类型：
--
作者：
Pok, Sharon;Wen, Victoria;Teoh, Narci C.
背景和目的：通过阵列-比较基因组杂交，我们证明了细胞周期蛋白E是Ku70 DNA修复缺陷小鼠中与肝细胞癌（HCC）发展相关的七个基因之一。因此，我们探索了在肝癌发生过程中，细胞周期蛋白E激酶可以克服p53的抑制作用的假设，并确定异常的miRNA(mi-R)-34，细胞周期蛋白E和p53的共同调节因子，是否可以解释它们作为HCC的“驱动因素”的相互作用。方法：用二乙基亚硝胺（DEN）注射C57BL/6雄性小鼠，在3-12月龄时产生发育不良肝细胞（dn）和hcc。结果：Cyclin 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，并定位于激光解剖的谷胱甘肽- s转移酶pi型阳性细胞。在DNs和hcc中，尽管p53和p21表达最多，但细胞周期蛋白E激酶活性先于细胞周期蛋白D1、增殖细胞核抗原表达。通过免疫沉淀实验，我们证实了细胞周期蛋白E，而不是细胞周期蛋白D1，是肝癌发生的增殖驱动因素。实验显示p21优先结合细胞周期蛋白D1，允许细胞周期蛋白E介导的从G1/S检查点“逃逸”。然后，我们在原发性HCC细胞中通过敲低实验证明cyclin E负责调节野生型p53；cyclin e敲低可增加p53和p21，降低抗凋亡Bcl-XL，降低细胞活力。相反，阻断p53可增强cyclin E、Bcl-XL的表达并增加细胞增殖。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，miR-34a、c在发育不良小鼠、人肝脏和hcc中表达上调，并且似乎与细胞周期蛋白E/p53有关。结论：通过miR34表达的功能活性细胞周期蛋白E的上调与p53功能的缺失与细胞周期检查点失败增加增殖驱动相关，从而促进肝癌的发生。
Background and Aim: 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. Methods: Dysplastic hepatocytes (DNs) and HCCs were generated from diethylnitrosamine (DEN)-injected C57BL/6 male mice at 3-12 months. Results: 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. Conclusion: 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.