• Journal of Microbiology
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• v.41 no.4
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• pp.300-305
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• 2003

Intracellular expression of RNA decoys, such as TAR or RRE decoy, has been previously shown to protect immune cells from human immunodeficiency virus type 1 (HIV-1) replication by inhibiting the binding of the HIV-1 regulatory protein to the authentic HIV RNA sequence. However, HIV-1 challenge experiments of primary human T cells, which express the RNA decoy, demonstrated that the cells were only transiently protected, and hence, more improved protocols for HIV-1 inhibition with the RNA decoys need to be developed. In this report, in order to develop a more effective RNA decoy, we analyzed and compared the ability of a series of RNA decoy derivatives in inhibiting HIV-1 replication in CEM cells. Using an improved tRNA cassette to express high levels of RNA decoy transcripts in cells, we found that co-expression of both TAR and RRE decoys, in the form of an aligned sequence in a single transcription cassette, much more potently blocked cells from HIV-1 than the expression of only one kind of RNA decoy. This observation will have an important implication for experiments involving optimization of clinical applications in RNA decoy-based gene therapy against HIV-1.

• Korean Journal of Microbiology
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• v.39 no.4
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• pp.235-241
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• 2003

We have previously demonstrated that intracellular expression of an RNA aptamer termed RRE40, which was selected in vitro to bind HIV Rev 10-fold much tighter than wild-type RRE, efficiently protected human CD4+ T cell line, CEM, from HIV-1. In this study, to evaluate the efficacy of the RRE40 RNA in clinical settings, polyclonal CD4+ peripheral blood lymphocytes (PBLs) were transduced with retroviral vectors expressing RRE40 decoy RNA and then challenged with clinical isolates of HIV-1. In contrast to the control cells transduced with vectors expressing control tRNA, intracellular expression of RRE40 RNA more effectively inhibited HIV-1 replication in CD4+ PBLs. However, transient and diminished inhibition, rather than complete inhibition, of HIV-1 replication in PBLs expressing RRE40 decoys have been observed. These results suggest that RRE40 decoy RNA would be useful to inhibit HIV-1 replication in cells. However, development of more efficient gene transfer protocols and/or more effective decoy RNAs would be needed to apply RNA decoy to modulate HIV-1 patient.

• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.707-713
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• 2000

Myasthenia gravis (MG) is caused mainly by autoantibodies directed against acetylcholine receptors located in the postsynaptic muscle cell membrane. Using in vitro selection techniques, we isolated an RNA containing 2'-fluoro pyrimidines that can specifically and avidly ($K_d$ ~25 nM) bind rat monoclonal antibody called mAb198, which recognizes the main immunogenic region on the acetylcholine receptors. This RNA can act as a very effective decoy and block mAb198 binding to the receptors in vitro. Furthermore, this RNA decoy can prevent the antigenic modulation of the acetylcholine receptor caused by mAb198 in human muscle cell cultures with and $IC_{50} $of approximately $2.4{\mu}M$. These results indicate that the RNA selected in this study is a more potent decly inhibitor of myashthenic antibodies than the previously identified RNA with 2'-amino pyrimidines [11]. Moreover, this RNA cross-reacts with autoantibodies from patients with MG and can protect human cells from the effects of these antibodies. These observations have important implications for developing an antigen-specific treatment of autoimmune diseases including MG, which is based on decoy RNAs selected in vitro.

• Archives of Plastic Surgery
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• v.33 no.1
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• pp.39-45
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• 2006

This study is to examine the relationship between TGF-b1 expression and CTGF expression, and to evaluate the effect of Sp1 blockade on the expression of TGF-b1, CTGF and extracellular genes, clones of fibroblasts stably transfected with Sp1 decoy ODN. R-Sp1 decoy ODN was highly resistant to degradation by nucleases or serum, compared to the linear or phosphorothioated-Sp1 decoy ODN. Skin wounds were created on the back of 36 anesthetized rats. They were divided into four groups-the rats with normal skin, with wounded skin without decoy, with wounded skin injected with R-Sp1 decoy, and with wounded skin injected with mismatched R-Sp1 decoy, respectively. Skins were collected at 3rd, 5th, 7th, 14th day after wounding. Cellular RNA was extracted by RT-PCR analysis. TGF-${\beta}1$ and CTGF were deeply related with skin fibrosis during scar formation and it appeared that TGF-${\beta}1$ may cause the induction of CTGF expression. R-Sp1 decoy ODN inhibited TGF-${\beta}1$ and CTGF expression both in cultured fibroblasts and in the skin of rats. These results indicate that targeting Sp1 with R-type decoy efficiently blocks extracellular matrix gene expression, and suggest an important new therapeutic approach to control the scarring in normal wound healing and fibrotic disorders.

• BMB Reports
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• v.50 no.4
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• pp.226-231
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• 2017

At the post-transcriptional and translational levels, microRNA (miRNA) represses protein-coding genes via seed pairing to the 3' untranslated regions (UTRs) of mRNA. Although working models of miRNA-mediated gene silencing are successfully established using miRNA transfections and knockouts, the regulatory interaction between miRNA and long non-coding RNA (lncRNA) remain unknown. In particular, how the mRNA-resembling lncRNAs with 5' cap, 3' poly(A)-tail, or coding features, are regulated by miRNA is yet to be examined. We therefore investigated the functional interaction between miRNAs and lncRNAs with/without those features, in miRNA-transfected early zebrafish embryos. We observed that the greatest determinants of the miRNA-mediated silencing of lncRNAs were the 5' cap and 3' poly(A)-tails in lncRNAs, at both the post-transcriptional and translational levels. The lncRNAs confirmed to contain 5' cap, 3' poly(A)-tail, and the canonical miRNA target sites, were observed to be repressed in the level of both RNA and ribosome-protected fragment, while those with the miRNA target sites and without 5' cap and 3' poly(A)-tail, were not robustly repressed by miRNA introduction, thus suggesting a role as a miRNA-decoy.

• Archives of Pharmacal Research
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• v.27 no.12
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• pp.1258-1262
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• 2004

Phosphodiesterase (PDE) 4 is an enzyme that degrades intracellular cAMP. In the present study, the effect of rolipram, a specific phosphodiesterase (PDE) 4 inhibitor, on osteoclast formation was investigated. Rolipram induced osteoclast formation in cocultures of mouse bone marrow cells and calvarial osteoblasts. This activity was not observed in the absence of calvarial osteoblasts, suggesting that calvarial osteoblasts are likely target cells of rolipram. Osteoclast formation by rolipram was completely blocked by the addition of osteoprotegerin (OPG), a soluble decoy receptor for the osteoclast differentiation factor, TNF-related activation-induced cytokine (TRANCE, identical to RANKL, ODF, and OPGL). Northern blot analysis revealed the effect of rolipram to be associated with the increased expression of TRANCE mRNA in mouse calvarial osteoblasts. Collectively, these data indicate that PDE4 inhibitor up-regulates the TRANCE mRNA expression in osteoblasts, which in turn controls osteoclast formation.

• YAKHAK HOEJI
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• v.48 no.3
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• pp.197-201
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• 2004

Phosphodiesterases (PDEs) are enzymes that degrade intracellular cAMP. In the present study, pentoxifylline, a PDE inhibitor, induced osteoclast formation in co-cultures of mouse bone marrow cells and calvarial osteoblasts. To address the involvement of the osteoclast differentiation factor TNF-related activation-induced cytokine (TRANCE, identical to RANKL, ODF, and OPGL), mouse bone marrow cells and calvarial osteoblasts were co-cultured with pentoxifylline in the presence of OPG, a decoy receptor for TRANCE. The osteoclastogenic effect of pentoxifylline was completely blocked by addition of OPG, suggesting that TRANCE is involved in the osteoclast formation induced by pentoxifylline, Northern blot analysis revealed that pentoxifylline significantly induced TRANCE mRNA expression in calvarial osteoblasts. These results suggests that pentoxifylline regulates TRANCE expression in osteoblasts, which in turn controls osteoclast formation.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.2
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• pp.719-724
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• 2012

This study aimed to investigate the clinical significance of expression and amplification of decoy receptor 3 (DcR3) in pancreatic carcinomas (PC). mRNA expression was detected by PQ-PCR, and amplification was determined. DcR3 protein expression was detected by immunohistochemistry and ELISA. Correlations between DcR3 expression and clinical pathological factors were analyzed. The relative amount of DcR3 in PC tissues and non-cancerous tissues showed a statistically significant difference, 21 cases displaying more than two fold DcR3 amplification, while no such amplification was found in normal pancreatic tissues. DcR3 positive cell staining was located in the cytoplasm. The positive rate of DcR3 in PC and non-cancerous tissues showed a significant difference. DcR3 mRNA expression was correlated with clinical staging, size of the tumor, lymph node metastasis and histological staging, while protein expression was correlated with clinical data like tumor size. DcR3 gene amplification only correlated with tumor size. The level of DcR3 in serum of the PC resectable group before operation was $72.2{\pm}10.2$ pg/ml, showing a significant difference compared to gallbladder carcinoma group (GC) or pancreatic benign tumor (PBT) group (P < 0.01). In conclusion, DcR3 amplification is correlated with DcR3 expression in PC tissues, especially those clinical pathological factors which reflect tumor progression. Assessment of DcR3 level in sera of PC patients may be helpful for the early diagnosis and prognostic judgement.

• Journal of Periodontal and Implant Science
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• v.32 no.4
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• pp.733-744
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• 2002

The fibroblasts are the principal cells in the periodontal ligament of peridontium. As the periodontal ligament fibroblasts (PDLF) show similar phenotype with osteoblasts, the PDLF are thought to play an important role in alveolar bone remodeling. Cell-to-cell contacted signaling is crucial for osteoclast formation. Recently it has been reported that PDLJ enhance the bone resorbing activity of osteoclasts differentiated from hematopoietic preosteoclasts. The aims of this study were to $clarify\;^{1)}$ the mechanism of PDLF-induced osteoclastogenesis $and\;^{2)}$ whether we can use preosteoclast cell line instead of primary hematopoietic preosteoclast cells for studying the mechanism of PDLF-induced osteoclastogenesis. Osteoclastic differentiation of mouse macrophage cell line RAW264.7 was compared with that of mouse bone marrow-derived M-CSF dependent cell (MDBM), a well-known hematopoietic preosteoclast model, by examining, 1) osteoclast-specific gene expression such as calcitonin receptor, M-CSF receptor (c-fms), cathepsin K, receptoractivator nuclear factor kappa B (RANK) ,2) generation of TRAP(+) multinucleated cells (MNCs), and 3) generation of resorption pit on the $OAAS^{TM}$ plate. RAW264.7 cultured in the medium containing of soluble osteoclast differentiation Factor (sODF) showed similar phenotype with MDBM-derived osteoclasts, those are mRNA expression pattern of osteoclast-specific genes, TRAP(+) MNCs generation, and bone resorbing abivity. Formation of resorption pits by osteoclastic MNCs differentiated from sODF-treated RAW264.7, was completely blocked by the addition of osteoprotegerin (OPG), a soluble decoy receptor for ODF, to the sODF-containing culture me야um. The effects of PDLF on differentiation of RAW264.7 into　the TRAP(+) multinucleated osteoclast-like cells were examined using coculture system. PDLF were fxed with paraformaldehyde, followed by coculture with RAW264.7, which induced formation of TRAP(+) MNCs in the absence of additional treatment of sODF. When compared with untreated and fixed PDLF (fPDLF), IL-1 ${\beta}$-treated, or lipopolysaccha-ride-treated and then fixed PDLF showed two-folld increase in the supporting activity of osteoclastogenesis from RAW264.7 coculture system. There were no TRAP(+) MNCs formation in coculture system of RAW264.7 with PDLF of no fixation. These findigs suggested that we can replace the primary hematopoietic preosteoclasts for RAW264. 7 cell line for studying the mechanism of PDLF-induced osteoclastogenesis, and we hypothesize that PDLF control osteoclastogenesis through ODF expression which might be enhanced by inflammatory signals.