• International Journal of Oral Biology
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• v.38 no.4
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• pp.155-160
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• 2013

The major issue in the development of nucleic acid based therapeutics is the inefficient delivery of these agents into cells. We prepared cholesterol conjugated spermine and evaluated its usefulness as a delivery modality for antisense oligonucleotides in HeLa-Luc cells. A 2'-O-methyl antisense oligonucleotide sequence, designed to correct splicing at an aberrant intron inserted into a normal luciferase reporter gene, was used for complex formation with cholesterol conjugated spermine. Effective delivery of this antisense agent into nucleus would results in the expression of a luciferasereporter gene product. The cholesterol-spermine formed stable complexes with the antisense oligonucleotide and showed modest delivery activity. Furthermore, this delivery activity was maintained even in the presence of serum proteins, mimicking in vivo conditions. Cholesterol-spermine thus has potential as a delivery system for antisense oligonucleotides into cells.

• Archives of Pharmacal Research
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• v.20 no.5
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• pp.438-442
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• 1997

Antisense oligonucleotide represents an interesting tool for selective inhibition of gene expression. However, their low efficiency of introduction within intact cells remains to be overcome. Antisense-$TGF{\beta}$ (25 mer) and antisense-$TGF{\beta}$ (18 mer) were used to study the cellular transport and biological function of antisense oligonucleotide in vitro. Since TGF and TNF play on important role in regulating the nitric oxide production from macrophages, the action of the above antisense oligonucleotides was easily monitored by the determination of nitrite. Poly-L-lysine, benzalkonium chloride and tetraphenylphosphonium chloride were used as polycations, which neutralize the negative charge of antisense oligonucleotide. The production of nitric oxide mediated by .gamma.-IFN in mouse peritoneal macrophage was increased by antisense-TGF.betha. in a dose-dependent manner. Antisense-$TGF{\beta}$ reduced the nitric oxide release from activated RAW 264.7 cells. Significant enhancement in the nitric oxide production was investigated by the cotreatment of poly-L-lysine with antisense-$TGF{\beta}$On the meanwhile, inhibition effect of antisense-$TGF{\beta}$ is not changed by the addition of poly-L-lysine. These results demonstrate that control of expression of $TGF{\beta}$ and TNF.alpha. gene is achieved using antisense technology and the cellular uptake of antisense oligonucleotide could be enhanced by ion-pairing.

• Korean Journal of Microbiology
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• v.36 no.2
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• pp.103-108
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• 2000

We have cloned and analyzed cDNA coding for non-virion (NV) protein of the m V - P R T The NV gene contained 336 bp open readmg frame and encoded a protein of 11 1 amino acids with a molecular weight of 13.2 kDa. The deduced amino acid sequence of NV of IHNVPRT was found to be 90-95% identical to those of foreign isolates of IHNV. These results indicate that NV gene of the MNV is highly conserved among &ifferent strains of THNV Northern blot analyses revealed that the levels of NV gene expression were strongly elevated after 20 h post-infection. In order to identify the role of NV in the replication of MNV in fish cells, IHNVinfected cells were treated with antisense oligonucleotides. While IHNV-PRT exposed to glycoprotein (G) antisense oligonucleotide showed severely reduced growth, the growth of virus exposed to NV antisense oligonucleotide was not affected by NV antisense oligonucleotide, which suggests that NV is not essential for replication of IHNV in fish cells.

• Molecules and Cells
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• v.46 no.1
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• pp.10-20
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• 2023

Antisense oligonucleotide (ASO) technology has become an attractive therapeutic modality for various diseases, including Mendelian disorders. ASOs can modulate the expression of a target gene by promoting mRNA degradation or changing pre-mRNA splicing, nonsense-mediated mRNA decay, or translation. Advances in medicinal chemistry and a deeper understanding of post-transcriptional mechanisms have led to the approval of several ASO drugs for diseases that had long lacked therapeutic options. For instance, an ASO drug called nusinersen became the first approved drug for spinal muscular atrophy, improving survival and the overall disease course. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF). Although Trikafta and other CFTR-modulation therapies benefit most CF patients, there is a significant unmet therapeutic need for a subset of CF patients. In this review, we introduce ASO therapies and their mechanisms of action, describe the opportunities and challenges for ASO therapeutics for CF, and discuss the current state and prospects of ASO therapies for CF.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.5
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• pp.2235-2240
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• 2012

c-Src is one member of non-receptor tyrosine kinase protein family that has over expression and activation in many human cancer cells. It has been shown that c-Src is implicated in various downstream signaling pathways associated with EGFR-dependent signaling such as MAPK and STAT5 pathways. Transactivation of EGFR by c-Src is more effective than EGFR ligands. To inhibit the c-Src expression, we used c-Src antisense oligonucleotide complexed with PAMAM Denderimes. The effect of c-Src antisense oligonucleotide on HT29 cell proliferation was determined by MTT assay. Then, the expression of c-Src, EGFR and the genes related to EGFR-depended signaling with P53 was applied by real time PCR. We used western blot analysis to elucidate the effect of antisense on the level of c-Src protein expression. The results showed, c-Src antisense complexed with PAMAM denderimers has an effective role in decrease of c-Src expression and EGFR-dependent downstream genes.

• Journal of Applied Biological Chemistry
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• v.44 no.4
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• pp.163-166
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• 2001

Piezoelectric (PZ) crystal biosensor system was used to detect the DNA of food pathogenic Listeria monocytogenes. L. monocytogenes-specific DNA was multiplied via the polymerase chain reaction using LM1 oligonucleotide (5'-TTACGAATTAAAAAGGAGCG-3') and LM2 oligonucleotide (5'-TTAAATCAGCAGGGGTCTTT-3') as primers. DNA fragment of 161 bp, which was specific only for L. monocytogenes, was observed. To obtain a large amount of single-stranded DNA containing an SH group used for coupling to the gold electrode chemisorptively, LM1 oligonucleotide containing a mercaptohexyl group was utilized as a single strand PCR primer. The PCR product was immobilized onto the gold electrode of PZ crystal, and hybridization was monitored in quartz crystal microbalance (QCM) system by injecting the antisense single-stranded DNA of 161 nucleotides obtained via the single strand PCR using the unmodified LM2 primer. Approximately 70 Hz of frequency drop was observed in the QCM system in the case of two consecutive injections of $5{\mu}g$ of the antisense single-stranded DNA.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.29 no.2
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• pp.86-94
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• 2003

It has been known that transglutaminase C (TGase C, TGase II) is directly participated in the DNA organization of chromosome, and affects the cellular processes such as proliferation, differentiation, and apoptosis of cells, but still not known what mechanism is working on. In this study, the cytogenetic and the immunohistochemical methods were used to observe the TGase C expression in the nuclear chromosome of the proliferating cells, especially in mitotic stage. The human gastric adenocarcinoma (SNU-1) cell line was used for immunohistochemistry and antisense inhibition study in vitro. The present study was also aimed to disclose the efficiency of antisense inhibition by using antisense oligonucleotide DNA labeled with fluorescence, and found that anti-TGase C probe was diffusely infiltrated into the cytoplasm and the nucleus of the cell. By the antisense inhibition the nuclei of SNU-1 cells became rough nuclear shape, as they were greatly reduced in TGase C immunoreactivity both for the normal and apoptotic SNU-1 cells. However, it is clearly presumed that the TGase C directly interacts with the chromosome of SNU-1 cells and it may play an important role in the division and organization of the chromosome during the mitotic stage.

• The Korean Journal of Physiology and Pharmacology
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• v.12 no.1
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• pp.1-6
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• 2008

Ribbon-type antisense oligonucleotide to TGF-${\beta}1$ (TGF-${\beta}1$ RiAS) was designed and tested to prevent or resolve the fibrotic changes induced by $CCl_4$ injection. When Hepa1c1c7 cells were transfected with TGF-${\beta}1$ RiAS, the level of TGF-${\beta}1$ mRNA was effectively reduced. TGF-${\beta}1$ RiAS, mismatched RiAS, and normal saline were each injected to mice via tail veins. When examined for the biochemical effects on the liver, TGF-${\beta}1$ mRNA levels were significantly reduced only in the TGF-${\beta}1$ RiAS-treated group. The results of immunohistochemical studies showed that TGF-${\beta}1$ RiAS prevented the accumulation of collagen and ${\alpha}$-smooth muscle actin, but could not resolve established fibrosis. These results indicate that ribbon antisense to TGF-${\beta}1$ with efficient uptake can effectively prevent fibrosis of the liver.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.4
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• pp.2361-2366
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• 2013

Diverse studies have shown that miR-155 is overexpressed in different tumor types. However, the precise molecular mechanism of the ectopic expression of miR-155 in breast cancer is still poorly understood. To further explore the role of miR-155 in breast tumorigenesis, we here assessed the influence of miR-155 antisense oligonucleotide (miR-155 ASO) on MDA-MB-157 cell viability and apoptosis in vitro. Furthermore, the effects of inhibitory effects of miR-155 on the growth of xenograft tumors in vivo were determined with performance of immunohistochemistry to detect expression of caspase-3, a pivotal apoptosis regulatory factor, in xenografts. Transfection efficiency detected by laser confocal microscope was higher than 80%. The level of miR-155 expression was significantly decreased (P<0.05) in the cells transfected with miR-155 ASO, compared with that in cells transfected with a negative control. After being transfected with miR-155 ASO, the viability of MDA-MB-157 cells was reduced greatly (P<0.05) and the number of apoptotic cells was increased significantly. Additionally, miR-155 ASO inhibited the growth of transplanted tumor in vivo and significantly increased the expression of caspase-3. Taken together, our study revealed that miR-155 ASO can induce cell apoptosis and inhibit cell proliferation in vitro. Moreover, miR-155 ASO could significantly repress tumor growth in vivo, presumably by inducing apoptosis via caspase-3 up-regulation. These findings provide experimental evidence for using miR-155 as a therapeutic target of breast carcinoma.

• Toxicological Research
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• v.18 no.4
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• pp.411-416
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• 2002

Apoptosis, or programmed cell death, is a genetically regulated pathway that is altered in many cancers. Overexpression of bcl-2 leads to resistance to apoptosis and promotes tumorigenesis. To determine the effect of bcl-2 antisense treatment in human lung cancer cell lines, a 20 mer full phosphorothioate oligonucleotide (ODN) targeted at the coding region of the bcl-2 mRNA was synthesized. Western blot analyses were used to examine bcl-2 protein level in five human non-small cell lung cancer (NSCLC) cell lines (NCI-H226, SK-MES-1 NCI-H358, NCI-H522 and NCI-Hl 299) and four human small cell lung cancer (SCLC) cell lines (NCI-H69, NCI-H4l7, HCC-2108 and SW2). Three out of five NSCLC (NCI-H226, SK-MES-1 and NCI-Hl 299) and all of SCLC cell lines expressed Bcl-2 protein. Treatment of these cell with antisense ODN for 48 hours reduced their viability and Bcl-2 protein level. As a conclusion, bcl-2 antisense treatment appears reduction of the Bcl-2 protein levels and cytotoxic effect including apoptosis in human lung cancer cell lines.