Dipartimento di Scienze per la Promozione della Salute "G. D'Alessandro," Sezione di Farmacologia "P. Benigno," Università degli Studi di Palermo, Palermo, Italy.
OMICS. 2012 Nov;16(11):579-88. doi: 10.1089/omi.2012.0048. Epub 2012 Oct 24.
Abstract Raf-1 kinase inhibitor protein (RKIP) is a tumor and metastasis suppressor that promotes drug-induced apoptosis in cancer cells. It is frequently downregulated, both at the mRNA and protein level, in hepatocellular carcinoma (HCC), but the mechanisms leading to this reduction are obscure. We sequenced the whole RKIP gene in three human HCC cell lines (HA22T/VGH, HepG2, and Hep3B), and in five clinical HCC samples, but could not find any gene variant that might account for their low RKIP levels. We also examined whether gene methylation may be responsible for the altered RKIP expression. No methylation of the RKIP gene was found in the tumor samples, while among the cell lines only Hep3B showed methylation of the gene, which was reduced by treatment with 5-aza-2'-deoxycytidine (5-AZA). The same treatment caused upregulation of RKIP at the mRNA, but not at the protein level, indicating that gene methylation is not a principal mechanism of the decrease in RKIP in the Hep3B cells. Furthermore, different elements consistently suggested that RKIP may be a target repressed by miR-224, a miRNA that is frequently and specifically upregulated in HCC, but our results excluded that this occurs, at least in the HCC cell lines. Factors like Snail, EZH2, and HDAC, have been implicated in the RKIP downregulation present in breast and prostate tumors, though some of our results from the cell lines do not support that they play such a role in HCC; however, this aspect is worthy of further study. However, recent results of ours and others suggest a significant involvement of proteosomal degradation and of its pharmacological inhibition. In conclusion, the causes of RKIP downregulation in HCC remain incompletely understood. However, we think that the present observations will be useful to generate further research, with the ultimate possible goal of devising specific approaches to restore the relevant antitumor function of the factor.
摘要 Raf-1 激酶抑制剂蛋白（RKIP）是一种肿瘤和转移抑制因子，可促进癌细胞中药物诱导的细胞凋亡。在肝细胞癌（HCC）中，RKIP 的 mRNA 和蛋白水平经常下调，但导致这种下调的机制尚不清楚。我们对三种人 HCC 细胞系（HA22T/VGH、HepG2 和 Hep3B）和五个临床 HCC 样本进行了 RKIP 全基因测序，但未能发现任何可能解释其低 RKIP 水平的基因变异。我们还研究了基因甲基化是否可能是导致 RKIP 表达改变的原因。在肿瘤样本中未发现 RKIP 基因甲基化，而在细胞系中仅 Hep3B 显示基因甲基化，5-氮杂-2'-脱氧胞苷（5-AZA）处理可降低基因甲基化。相同的处理导致 RKIP 在 mRNA 水平上调，但在蛋白质水平没有上调，表明基因甲基化不是 Hep3B 细胞中 RKIP 减少的主要机制。此外，不同的因素表明 RKIP 可能是 miR-224 抑制的靶标，miR-224 是 HCC 中经常且特异性上调的 miRNA，但我们的结果排除了这种情况发生，至少在 HCC 细胞系中是这样。Snail、EZH2 和 HDAC 等因子已被牵连到乳腺癌和前列腺癌中 RKIP 的下调，但我们从细胞系得到的一些结果并不支持它们在 HCC 中发挥这样的作用；然而，这一方面值得进一步研究。然而，我们和其他人的最新研究结果表明，蛋白体降解及其药理学抑制的显著参与。总之，HCC 中 RKIP 下调的原因仍不完全清楚。然而，我们认为目前的观察结果将有助于进一步研究，最终可能会制定出恢复该因子相关抗肿瘤功能的具体方法。
