State Key Laboratory of Infectious Disease Diagnosis and Treatment, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, P.R. China.
Oncol Rep. 2010 May;23(5):1457-62. doi: 10.3892/or_00000784.
Dysregulation of the antiapoptotic protein cellular FLICE-like inhibitory protein (c-FLIP) has been proven to be associated with tumorigenesis and progress of most human cancers. However, its aberrant expression is poorly elucidated. MicroRNAs (miRNAs) are small non-coding RNAs that are involved in tumorigenesis through negatively regulating gene expression. Our study disclosed that c-FLIP was overexpressed in HepG2 hepatocellular carcinoma cells and down-regulation of c-FLIP enhanced taxol-induced apoptosis. Taxol induction significantly decreased the protein level of c-FLIP. While no decrease in c-FLIP mRNA level was observed, indicating taxol decreased c-FLIP expression through a post-transcriptional mechanism. miR-512-3p was a predicted suppressor of c-FLIP and exhibited an opposite expression manner to c-FLIP before and after taxol induction. Luciferase report assay demonstrated miR-512-3p negatively regulated c-FLIP expression via a conserved miRNA-binding site in 3' untranslated region (3'UTR) of c-FLIP. The decrease of c-FLIP protein due to transfection of miR-512-3p further validated the inhibitory effect of miR-512-3p on c-FLIP. Additional transfection of miR-512-3p remarkably promoted taxol-induced apoptosis, confirming its involvement in apoptosis. In summary, our study disclosed a novel regulatory mechanism that down-regulation of c-FLIP by miR-512-3p contributed to taxol-induced apoptosis. Importantly, the pivotal role of miR-512-3p in determining c-FLIP abundance helps to broaden the implications for cancer therapy by developing small molecules to directly target c-FLIP at mRNA level.
细胞型 Fas 相关死亡结构域蛋白抑制蛋白（c-FLIP）的抗凋亡蛋白的失调已被证明与大多数人类癌症的肿瘤发生和进展有关。然而，其异常表达的机制仍不清楚。microRNAs（miRNAs）是一种小的非编码 RNA，通过负调控基因表达参与肿瘤发生。我们的研究表明，c-FLIP 在 HepG2 肝癌细胞中过表达，下调 c-FLIP 可增强紫杉醇诱导的细胞凋亡。紫杉醇诱导显著降低 c-FLIP 的蛋白水平。然而，c-FLIP mRNA 水平没有下降，表明紫杉醇通过转录后机制降低 c-FLIP 的表达。miR-512-3p 是 c-FLIP 的预测抑制子，在紫杉醇诱导前后表现出与 c-FLIP 相反的表达模式。荧光素酶报告实验表明，miR-512-3p 通过 c-FLIP 3'UTR 中的保守 miRNA 结合位点负调控 c-FLIP 的表达。miR-512-3p 转染导致 c-FLIP 蛋白减少，进一步验证了 miR-512-3p 对 c-FLIP 的抑制作用。转染 miR-512-3p 后 c-FLIP 蛋白进一步减少，进一步验证了 miR-512-3p 对 c-FLIP 的抑制作用。miR-512-3p 的额外转染显著促进了紫杉醇诱导的细胞凋亡，证实了其在凋亡中的作用。总之，我们的研究揭示了一种新的调控机制，即 miR-512-3p 下调 c-FLIP 有助于紫杉醇诱导的细胞凋亡。重要的是，miR-512-3p 在决定 c-FLIP 丰度方面的关键作用有助于通过开发直接靶向 mRNA 水平的 c-FLIP 的小分子来拓宽癌症治疗的应用。
