Abstract
BackgroundSince 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 FindingsBy 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/SignificanceTogether, 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.
Highlights
A malignant tumor is caused by a series of abnormal expressions and/or deviant functions of genes governing cell proliferation and differentiation
We show that CK18 filaments are reorganized in the Cl1 cells; a portion of the Cl1 cell population is delayed at the S and G2/M phases of their cell cycle; and the average phosphorylated CK18 and the total amount of CK18 are lower in Cl1 than in 7721
We propose that the inhibition of the protein kinase Ce (PKCe) activity, and the suppression of the SMMC-7721 hepatoma cell growth, was achieved though the formation of a complex by direct physical interaction between C/EBPb 39untranslated region (39UTR) RNA, PKCe and CK18
Summary
A malignant tumor is caused by a series of abnormal expressions and/or deviant functions of genes governing cell proliferation and differentiation (including proto-oncogenes and tumor suppressor genes). The protein kinase Ce (PKCe) is an oncogene important in tumorigenesis [1,2]. PKCe has been classified as a novel PKC isotype and is characterized as calcium-independent and phorbol ester/diacylglycerol-sensitive. A characteristic of PKCe is that it binds a large number of interacting proteins, indicating the generality of its actions. It is activated in the cytoplasm by diacylglycerol or phorbol esters, and it phosphorylates downstream target molecules, thereby transducing growth signals into the nucleus to promote gene expression [3]. We sought to determine the molecular mechanism of the tumor suppression activity of a short RNA from 39UTR of C/EBPb mRNA (C/EBPb 39UTR RNA) in human hepatocarcinoma cells SMMC-7721
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