Department of Medical, Technological and Translational Sciences, University Hospital of Cattinara, Trieste, Italy.
Dig Liver Dis. 2011 Dec;43(12):1006-14. doi: 10.1016/j.dld.2011.07.007. Epub 2011 Aug 9.
No effective therapy is available for hepatocellular carcinoma. To identify novel therapeutic strategies, we explored the effects of the depletion of E2F1, cyclin E1-E2 whose inter-relationships in hepatocellular carcinoma cell proliferation have never been defined.
siRNA-mediated depletion of the targets was studied in the hepatocellular carcinoma cells HepG2, HuH7 and JHH6 characterized by high, medium and low hepatocyte differentiation grade, respectively; a model of normal human hepatocytes was also considered.
The depletion of each target mRNA reduced the levels of the other two mRNAs, thus demonstrating a close regulatory control, also confirmed by over-expression experiments. At the protein level, an exception to this trend was observed for cyclinE1 whose amount increased upon cyclin E2 (HepG2, HuH7, JHH6) and E2F1 (HepG2) depletion. In HepG2, reduced cyclinE1 proteolysis accounted for this observation. Additionally, cyclin E1-E2-E2F1 targeting decreased the levels of cyclin A2 mRNA and of the hyper-phosphorylated form of pRb thus leading to an S-phase cell decrease; migration was impaired as well. Finally, the model of human hepatocytes used was clearly less affected by target mRNAs depletion than hepatocellular carcinoma cells.
Our data provide novel mutual relationships amongst cyclin E1-E2-E2F1 and indicate their role in sustaining hepatocellular carcinoma cell proliferation/migration, validating the concept of an anti-cyclin E1-E2-E2F1 therapeutic approach for hepatocellular carcinoma.
目前尚无有效的治疗方法用于肝细胞癌。为了寻找新的治疗策略，我们研究了 E2F1 和 cyclin E1-E2 的耗竭对肝细胞癌细胞增殖的影响，因为这两者之间的关系在肝细胞癌中尚未被定义。
在 HepG2、HuH7 和 JHH6 三种肝癌细胞系（分别具有高、中、低肝细胞分化程度）以及正常人类肝细胞模型中，采用 siRNA 介导的靶基因耗竭方法研究这些靶基因。
每种靶基因 mRNA 的耗竭均降低了另外两种 mRNA 的水平，这表明存在密切的调控控制，这一结果也通过过表达实验得到了证实。在蛋白水平上，这种趋势存在一个例外，即 cyclinE1 的量在 cyclin E2（HepG2、HuH7、JHH6）和 E2F1（HepG2）耗竭时增加。在 HepG2 中，cyclinE1 蛋白水解减少导致了这种观察结果。此外，cyclin E1-E2-E2F1 靶向作用降低了 cyclin A2 mRNA 的水平和 pRb 的高度磷酸化形式，从而导致 S 期细胞减少；迁移也受到损害。最后，所使用的人肝细胞模型比肝癌细胞受靶基因 mRNA 耗竭的影响明显更小。
我们的数据提供了 cyclin E1-E2-E2F1 之间新的相互关系，并表明它们在维持肝细胞癌细胞增殖/迁移中的作用，验证了针对肝细胞癌的抗 cyclin E1-E2-E2F1 治疗方法的概念。
