State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
PLoS One. 2011 Jan 24;6(1):e16543. doi: 10.1371/journal.pone.0016543.
Since the end of last century, RNAs from the 3'untranslated region (3'UTR) of several eukaryotic mRNAs have been found to exert tumor suppression activity when introduced into malignant cells independent of their whole mRNAs. In this study, we sought to determine the molecular mechanism of the tumor suppression activity of a short RNA from 3'UTR of C/EBPβ mRΝΑ (C/EBPβ 3'UTR RNA) in human hepatocarcinoma cells SMMC-7721.
METHODOLOGY/PRINCIPAL FINDINGS: By using Western blotting, immunocytochemistry, molecular beacon, confocal microscopy, protein kinase inhibitors and in vitro kinase assays, we found that, in the C/EBPβ 3'UTR-transfectant cells of SMMC-7721, the overexpressed C/EBPβ 3'UTR RNA induced reorganization of keratin 18 by binding to this keratin; that the C/EBPβ 3'UTR RNA also reduced phosphorylation and expression of keratin 18; and that the enzyme responsible for phosphorylating keratin 18 is protein kinase Cε. We then found that the C/EBPβ 3'UTR RNA directly inhibited the phosphorylating activity of protein kinase Cε; and that C/EBPβ 3'UTR RNA specifically bound with the protein kinase Cε-keratin 18 conjugate.
CONCLUSION/SIGNIFICANCE: Together, these facts suggest that the tumor suppression in SMMC-7721 by C/EBPβ 3'UTR RNA is due to the inhibition of protein kinase Cε activity through direct physical interaction between C/EBPβ 3'UTR RNA and protein kinase Cε. These facts indicate that the 3'UTR of some eukaryotic mRNAs may function as regulators for genes other than their own.
自上个世纪末以来，人们发现几种真核生物 mRNA 的 3'非翻译区（3'UTR）中的 RNA 可以在不依赖其全长 mRNA 的情况下独立地进入恶性细胞，从而发挥肿瘤抑制活性。在这项研究中，我们试图确定 C/EBPβ mRNA 的 3'UTR 中的一段短 RNA（C/EBPβ 3'UTR RNA）在人肝癌细胞 SMMC-7721 中发挥肿瘤抑制活性的分子机制。
方法/主要发现：通过 Western blot、免疫细胞化学、分子信标、共聚焦显微镜、蛋白激酶抑制剂和体外激酶测定，我们发现，在 SMMC-7721 的 C/EBPβ 3'UTR 转染细胞中，过表达的 C/EBPβ 3'UTR RNA 通过与角蛋白 18 结合诱导角蛋白 18 的重排；C/EBPβ 3'UTR RNA 还降低了角蛋白 18 的磷酸化和表达；负责磷酸化角蛋白 18 的酶是蛋白激酶 Cε。我们随后发现，C/EBPβ 3'UTR RNA 直接抑制蛋白激酶 Cε 的磷酸化活性；C/EBPβ 3'UTR RNA 特异性地与蛋白激酶 Cε-角蛋白 18 缀合物结合。
结论/意义：总之，这些事实表明，C/EBPβ 3'UTR RNA 在 SMMC-7721 中的肿瘤抑制作用是由于 C/EBPβ 3'UTR RNA 通过与蛋白激酶 Cε 的直接物理相互作用抑制蛋白激酶 Cε 的活性。这些事实表明，某些真核生物 mRNA 的 3'UTR 可能作为其自身以外的基因的调节剂发挥作用。
