Baiz Daniele, Dapas Barbara, Farra Rossella, Scaggiante Bruna, Pozzato Gabriele, Zanconati Fabrizio, Fiotti Nicola, Consoloni Lara, Chiaretti Sara, Grassi Gabriele
Daniele Baiz, Barbara Dapas, Bruna Scaggiante, Sara Chiaretti, Gabriele Grassi, Department of Life Sciences, University of Trieste, 34100 Trieste, Italy.
World J Gastroenterol. 2014 Jan 21;20(3):795-803. doi: 10.3748/wjg.v20.i3.795.
To evaluate the effects of the proteasome inhibitor bortezomib (BZB) on E2Fs and related genes in hepatocellular carcinoma (HCC) cells.
The mRNA levels of the E2F family members (pro-proliferative: E2F1-3 and anti-proliferative: E2F4-8) and of their related genes cyclins and cyclin-dependent kinases (cdks) were evaluated in two HCC cell lines following a single BZB administration. mRNA levels of the epithelial-mesenchymal transition (EMT) genes were also measured in both cell lines after BZB treatment. The BZB concentration (40 nmol/L) used was chosen to stay well below the maximal amount/cm² recommended for in vivo application, and 2 d incubation was chosen as this time point has been found optimal to detect BZB effects in our previous studies. The HCC cell lines, HepG2 and JHH6, were chosen as they display different phenotypes, hepatocyte-like for HepG2 and undifferentiated for JHH6, thus representing an in vitro model of low and high aggressive forms of HCC, respectively. The mRNA levels of the target genes were measured by two-color microarray-based gene expression analysis, performed according to Agilent Technologies protocol and using an Agilent Scan B. For the E2F family members, mRNA levels were quantified by real-time reverse transcription polymerase chain reaction (RT-PCR). Using small interfering RNA's, the effects of E2F8 depletion on cell number was also evaluated.
After BZB treatment, microarray analysis of the undifferentiated JHH6 revealed a significant decrease in the expression of the pro-proliferative E2F member E2F2. Quantitative RT-PCR data were in keeping with the microarray analysis, and showed a significant increase and decrease in E2F8 and E2F2 mRNA levels, respectively. In contrast, BZB treatment of the hepatocyte-like HCC cell line HepG2 had a significant impact on mRNA levels of 5 of the 8 E2F members. In particular, mRNA levels of the pro-proliferative E2F members E2F1, E2F2, and of the anti-proliferative member E2F8, decreased over 80%. Notably, a reduction in E2F8 expression in HepG2 and JHH6 cells following siRNA treatment had no impact on cell proliferation. As observed with JHH6, BZB treatment of HepG2 cells induced a significant increase in mRNA levels of an anti-proliferative E2F member, E2F6 in this case. As was observed with E2F's, more dramatic changes in mRNA levels of the E2F related genes cyclins and Cdks and EMT genes were observed after BZB treatment of HepG2 compared to JHH6.
The differential expression of E2Fs and related genes induced by BZB in diverse HCC cell phenotypes contribute to bortezomib's mechanism of action in hepatocellular carcinoma.
评估蛋白酶体抑制剂硼替佐米（BZB）对肝癌（HCC）细胞中E2F家族及相关基因的影响。
单次给予BZB后，评估两种肝癌细胞系中E2F家族成员（促增殖型：E2F1 - 3；抗增殖型：E2F4 - 8）及其相关基因细胞周期蛋白和细胞周期蛋白依赖性激酶（cdks）的mRNA水平。BZB处理后，还测量了两种细胞系中上皮 - 间质转化（EMT）基因的mRNA水平。所使用的BZB浓度（40 nmol/L）远低于体内应用推荐的最大量/cm²，选择2天的孵育时间是因为在我们之前的研究中发现该时间点最适合检测BZB的作用。选择肝癌细胞系HepG2和JHH6，因为它们表现出不同的表型，HepG2为肝细胞样，JHH6为未分化型，因此分别代表低侵袭性和高侵袭性肝癌的体外模型。通过基于双色微阵列的基因表达分析测量靶基因的mRNA水平，该分析按照安捷伦科技公司的方案进行，并使用安捷伦Scan B。对于E2F家族成员，通过实时逆转录聚合酶链反应（RT-PCR）对mRNA水平进行定量。使用小干扰RNA，还评估了E2F8缺失对细胞数量的影响。
BZB处理后，对未分化的JHH6进行微阵列分析显示，促增殖型E2F成员E2F2的表达显著降低。定量RT-PCR数据与微阵列分析结果一致，显示E2F8和E2F2的mRNA水平分别显著增加和降低。相比之下，对肝细胞样肝癌细胞系HepG2进行BZB处理对8个E2F成员中的5个成员的mRNA水平有显著影响。特别是，促增殖型E2F成员E2F1、E2F2以及抗增殖型成员E2F8的mRNA水平降低了80%以上。值得注意的是，siRNA处理后HepG2和JHH6细胞中E2F8表达的降低对细胞增殖没有影响。与JHH6一样，对HepG2细胞进行BZB处理诱导抗增殖型E2F成员E2F6的mRNA水平显著增加。与E2F家族成员情况相同，与JHH6相比，对HepG2进行BZB处理后，E2F相关基因细胞周期蛋白和Cdks以及EMT基因的mRNA水平变化更为显著。
BZB在不同肝癌细胞表型中诱导的E2F家族及相关基因的差异表达有助于硼替佐米在肝癌中的作用机制。
