MicroRNAs in human virus genomes: helping hands for viral infection.

MicroRNAs (miRNAs) are small noncoding regulatory RNAs that control essential cellular activities. Many viruses use these regulators to help infect host cells and maintain their presence in those cells. Therefore, virus-encoded miRNAs are important tools for viruses in the battle with their hosts. Knowledge of these miRNAs is essential for the development of new drugs to treat viral diseases and virus-associated cancers. In this minireview, the miRNAs encoded in viral genomes, their expression and functions, the interactions between viral and cellular miRNAs in viral diseases and virus- associated cancers, and the use of miRNAs for therapy are described.

Overexpression of hnRNPC2 induces multinucleation by repression of Aurora B in hepatocellular carcinoma cells.

Heterogeneous ribonuclear protein C2 (hnRNPC2), an RNA binding protein, is a component of hnRNPC which is upregulated in many tumors. Multinucleation exists in many tumors and is positively correlated with tumor grade. To uncover the correlation between hnRNPC2 and multi-nucleation in hepatocellular carcinoma SMMC-7721 cells, we constructed a pEGFP-hnRNPC2 vector and transfected it into cancer cells. Our results revealed that overexpression of hnRNPC2 induced multinucleation in SMMC-7721 cells. Tracking tests indicated that the induced multinucleated cells were unable to recover to mononuclear cells and finally died as a result of defects in cell division. Furthermore, Aurora B, which was localized at the midbody and plays a role in cytokinesis, was repressed in hnRNPC2-overexpressing cells, whose knockdown by RNA interference also induced multinucleation in SMMC-7721 cells. Quantitative polymerase chain reaction (qPCR) and mRNA-protein co-immunoprecipitation results revealed that Aurora B mRNA did not decrease in hnRNPC2-overexpressing cells, instead it bound more hnRNPC2 and less eIF4E, an mRNA cap binding protein and translational initiation factor. Moreover, hnRNPC2 bound more eIF4E in hnRNPC2-overexpressing cells. These results indicate that hnRNPC2 repressed Aurora B binding with eIF4F, which must bind with Aurora B mRNA in order to initiate its translation. This induced multinucleation in hepatocellular carcinoma cells. In addition, hnRNPC2 accelerated hepatocellular carcinoma cell proliferation. Collectively, these data suggest that hnRNPC2 may be a potential target for hepatocellular carcinoma cell diagnosis and treatment.

Cancer cell growth suppression by a 62nt AU-rich RNA from C/EBPß 3'UTR through competitive binding with HuR.

AU-rich elements are functional motifs in the 3'untranslated region of mRNA and are binding sites for the RNA binding protein HuR, an mRNA stabilizer and translation enhancer implicated in carcinogenesis. It is not clear whether, and, if so, how the AU-rich elements function in cells when they are separated from their mRNA and form an independent RNA species. Here, we show that a short RNA with AU-rich elements derived from C/EBPß 3'UTR suppressed growth in a human liver cancer cell line. It specifically bound HuR, and it competed with C/EBPß mRNA in order to bind to HuR. Our results provide evidence that the cancer cell growth suppression by this 62nt RNA containing AU-rich elements may be due to competitive binding to HuR. This work may open new options for the development of novel anti-cancer drugs.

Tumor suppression by RNA from C/EBPß 3'UTR through the inhibition of protein kinase Cε activity.

BACKGROUND: 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/EBPß mRΝΑ (C/EBPß 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/EBPß 3'UTR-transfectant cells of SMMC-7721, the overexpressed C/EBPß 3'UTR RNA induced reorganization of keratin 18 by binding to this keratin; that the C/EBPß 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/EBPß 3'UTR RNA directly inhibited the phosphorylating activity of protein kinase Cε; and that C/EBPß 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/EBPß 3'UTR RNA is due to the inhibition of protein kinase Cε activity through direct physical interaction between C/EBPß 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.

C/EBPbeta sustains undifferentiated state of murine embryonic stem cells as a mediator of LIF.

C/EBPbeta (also called NF-IL6) is a multifunctional transcription factor, a major function of which is the enhancement of cellular differentiation. Leukemia inhibitory factor (LIF) is a cytokine playing divergent roles in different cell types: induces differentiation in preadipocytes and, however, inhibits differentiation in pluripotent murine embryonic stem (mES) cells. However, roles of C/EBPbeta in mES cells are obscure. Here, we show for the first time that C/EBPbeta in the presence of LIF plays a role of sustaining undifferentiated state in this cell line, i.e. C/EBPbeta is a target of regulation of LIF, as described as follows. The expression of endogenous C/EBPbeta proteins in mES cells is positively correlated with the LIF added into medium. Even the exogenous C/EBPbeta proteins and their truncated form, LIP, artificially overexpressed in undifferentiated mES cells, do not enhance but inhibit ES cells' differentiation in the presence of LIF, and the long isoforms of C/EBPbeta proteins strongly enhance cell propagation. This enhancement lasts for a certain short time even after LIF removal. In the absence of LIF, C/EBPbeta and LIP induce expression of differentiation-related genes and promote mES cells' differentiation, as anticipated. With LIF, the expression levels of some differentiation-related genes regulated by C/EBPbeta and LIP were significantly lower than that without LIF. Therefore, in pluripotent mES cells C/EBPbeta is regulated by LIF and sustains undifferentiated state of those cells as a mediator of LIF.

Systemic delivery of full-length C/EBP beta/liposome complex suppresses growth of human colon cancer in nude mice.

C/EBP beta (CCAAT/enhancer-binding protein beta) is an important transcription factor involved in cellular proliferation and differentiation. Overexpression of the full-length C/EBP beta protein results in cellular growth arrest and apoptosis. Using a nonviral liposome as carrier, we delivered the full-length C/EBP beta expression plasmid, pCN, into nude mice bearing CW-2 human colon cancer tumors via tail vein. Southern blots revealed that the major organs and tumors were transfected. Experimental gene therapy showed that a strong suppression of tumor growth was observed in the pCN-treated mice, and such suppression was due to the overexpression of C/EBP beta, leading to the increased apoptosis in tumors of pCN-treated mice. No apparent toxic effects of pCN/liposome complex were observed in the animals. Thus, C/EBP beta has tumor suppression effect in vivo and may be used in gene therapy for cancers.

Direct isolation of specific RNA-interacting proteins using a novel affinity medium.

Isolation of proteins that specifically interact with a given RNA or RNA regulation element is essential for studies on the molecular mechanisms of gene expression. Here, a novel method for direct isolation of such interacting proteins is described. It uses an affinity medium that consists of an interacting RNA with an artificially added 'tail', which is annealed to one end of a DNA 'arm', the other end of which is fixed covalently on the surface of aminosilanized glass powder. Thus the RNA itself is fully suspending, facilitating its interactions with proteins in its natural conformation. The proteins bound on the interacting RNA are eluted and subjected to SDS-PAGE, and the Coomassie-stained protein bands are cut and subjected to mass spectrometry (MS) analysis. Using this method, three proteins specifically interacting with the C/EBPbeta 3'-untranslated region (3'-UTR) RNA were isolated and identified. This method is simple and convenient, and the DNA-glass powder medium can be used repeatedly.

Proteomic analysis of differential protein expression in a human hepatoma revertant cell line by using an improved two-dimensional electrophoresis procedure combined with matrix assisted laser desorption/ionization-time of flight-mass spectrometry.

The proteomic profiles of a human hepatoma revertant, CL1, and its original cell line, SMMC7721, were compared by using an improved two-dimensional electrophoresis (2-DE) procedure, with multi-IPGstrips gels (length

Multi-strips on one gel method to improve the reproducibility, resolution power and high-throughput of two-dimensional electrophoresis.

Two-dimensional polyacrylamide gel electrophoresis is one of the most key separation tools which can reveal hundreds or even thousands of proteins at a time in proteomic research. In this paper, we report a new IPG strip application, called multi-strips on one gel (MSOG) method. By comparing the 2-DE patterns of the same sample, the different state samples and the same sample in the different second dimensional SDS running systems (large size and medium size gels), we found this new method can not only improve the reproducibility and resolution power of 2-DE pattern, but also achieve high throughput and economical format which is helpful to automatic proteomic research.

Low content of protein S29 in ribosomes of human lung cancer cell line a549: detected by two-dimensional electrophoresis.

This study revealed that the content of protein S29 in ribosomes of cancer cell line A549 was distinctly low (equivalent to about 30% of that of 2BS). The conclusion was acquired based on the ratios of spot volume of ribosomal protein S29 to that of several other ribosomal proteins (S29/L37a, S29/L38, S29/S27 and S29/S28) in the same gel plate. The possible biological roles of ribosomal protein S29 in malignant transformation and translation regulation are briefly discussed.

Gene expression profile favoring phenotypic reversion: a clue for mechanism of tumor suppression by NF-IL6 3'UTR.

Transfection of cDNA in 3'untranslated region of human nuclear factor for interleukin-6 (NF-IL6 3'UTR) induced tumor suppression in a human hepatoma cell line. cDNA array analysis was used to reveal changes in gene expression profile leading to tumor suppression The results indicate that this suppression was not due to activation of dsRNA-dependent protein kinase, nor to inactivation of oncogenes; rather, all the changes in expression of known genes, induced by NF-IL6 3'UTR cDNA may be ascribed to the suppression of cellular malignancy. Therefore, our results imply that this 3'untranslated region may have played role of a regulator of gene expression profile.

Enhancement of Macrophage Cytotoxicity by Overexpression of Exogenous NF-IL6 Gene.

NF-IL6 (Nuclear factor for IL-6 expression) is involved in inflammatory reaction, expression of acute-phase proteins and cytokines, apoptosis and suppression of tumor cells, and maintenance of macrophage immunological functions. To investigate the role of highly expressed exogenous NF-IL6 in macrophage tumor cytotoxicity, a recombinant expression plasmid, pCN, which harbored the coding region of NF-IL6, was transfected into murine primary cultured peritoneal resident macrophages by an improved DEAE-dextran method. Western blot showed the high expression of NF-IL6 in these macrophages. Then the tumor cytotoxicity of the NF-IL6-overexpressing macrophages from normal and nude mice was measured by an alkaline phosphatase assay, using the human hepatocarcinoma cell line SMMC 7721 as target cells. Results showed that the overexpression of NF-IL6 enhanced the tumor cytotoxicity in both types of macrophages, demonstrating that the expression level of the NF-IL6 gene was directly related to the tumoricidal activity in these cells.

An NF-IL6 3'UTR RNA-specific Binding Protein in E. coli Purification and Partial Protein Sequencing.

protein was found in E.coli which can specifically bind to NF-IL6 mRNA 3'UTR. After a series of purification steps, the RNA-specific binding protein was directly sequenced on the Porton LF3200 Protein Sequencer. A sequence of 10 amino acids of the purified NF-IL6 3'UTR binding protein was obtained, namely, Ala-Thr-Arg-Ile-Glu-Phe-His-Gly-Cyss(?)-Gly. A BLAST search with the 10 amino acids against NCBI database failed to identify any protein with identical sequence. The significance of identifying an eukaryotic mRNA binding protein in E.coli is discussed.

On the Molecular Mechanism of the Tumor Suppressor Function of cDNA Clone p14-6.

Interactions between the RNA transcript of the tumor suppressor cDNA clone, p14-6 (the 3'untranslated region of the nuclear factor for human interleukin-6; NF-IL6 3'UTR), and the reversion-related proteins BNF, were investigated. It was found that: (1) the recognition site of the RNA for BNFs was a 24-nucleotide segment located within the 3'-proximal U-rich sequence; (2) the BNFs were a group of proteins which may interact with each other before interacting with a site on the RNA as a protein complex; (3) possibly only one protein in the complex, namelyR62, directly bound to the RNA site.