• Journal of Life Science
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• v.28 no.12
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• pp.1516-1522
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• 2018

Atherosclerosis is an obstructive vessel disease mainly caused by chronic arterial inflammation to which the proliferation and migration of vascular smooth muscle cells (VSMCs) is the main pathological response. In the present study, the primary responsible inflammatory cytokine and its signaling pathway was investigated. The proliferation and migration of VSMCs was significantly enhanced by the prostaglandin $F_{2{\alpha}}$ ($PGF_{2{\alpha}}$), while neither was affected by tumor necrosis factor ${\alpha}$. Prostacyclin $I_2$ was seen to enhance the proliferation of VSMCs while simultaneously suppressing their migration. Both prostaglandin $D_2$ and prostaglandin $E_2$ significantly enhanced the migration of VSMCs, however, proliferation was not affected by either of them. The proliferation and migration of VSMCs stimulated by $PGF_{2{\alpha}}$ progressed in a dose-dependent manner; the $EC_{50}$ value of both proliferation and migration was $0.1{\mu}M$. VSMCs highly expressed the phospholipase isoform $C-{\beta}3$ ($PLC-{\beta}3$) while others such as $PLC-{\beta}1$, $PLC-{\beta}2$, and $PLC-{\beta}4$ were not expressed. Inhibition of the PLCs by U73122 completely blocked the $PGF_{2{\alpha}}$-induced migration of VSMCs, and, in addition, silencing $PLC-{\beta}3$ significantly diminished the $PGF_{2{\alpha}}$-induced proliferation and migration of VSMCs. Given these results, we suggest that $PGF_{2{\alpha}}$ plays a crucial role in the proliferation and migration of VSMCs, and activation of $PLC-{\beta}3$ could be involved in their $PGF_{2{\alpha}}$-dependent migration.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.6
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• pp.2527-2532
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• 2012

Objectives: The aim of the present study was to explore mechanisms underlying the effects of down-regulating ${\beta}$-catenin expression on esophageal carcinoma (EC) cells. Methods: Cell cycle distribution and apoptosis were determined using flow cytometry and annexin V apoptosis assay, respectively. Transmission electron microscopy (TEM) was used to examine changes in ultrastructure, while expression of cyclin D1 protein and mRNA was detected by western blot and real-time PCR. Proliferating cell nuclear antigen (PCNA) and extracellular signal-regulated kinase (ERK) 1-2 were evaluated by Western blot analysis. PCNA labeling index (LI) was determined by immunocytochemistry. Results: Compared with pGen-3-con transfected and Eca-109 cells, the percentage of G0/G1-phase pGen-3-CTNNB1 transfected cells was obviously increased (P<0.05), with no significant difference among the three groups with regard to apoptosis (P>0.05). pGen-3-CTNNB1 transfected cells exhibited obvious decrease in cyclin D1 mRNA and protein expression (P<0.05) and the ultrastructure of Eca-109 cells underwent a significant change after being transfected with pGen-3-CTNNB1, suggesting that down-regulating ${\beta}$-catenin expression can promote the differentiation and maturation. The expression of PCNA and the ERKI/2 phosphorylation state were also down-regulated in pGen-3-CTNNB1 transfected cells (P<0.05). At the same time, the PCNA labeling index was decreased accordingly (P<0.05). Conclusion: Inhibition of EC Eca-109 cellproliferation by down-regulating ${\beta}$-catenin expression could improve cell ultrastructure by mediating blockade in G0/G1 through inhibiting cyclin D1, PCNA and the MAPK pathway (p-ERK1/2).

• Journal of Plant Biotechnology
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• v.36 no.1
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• pp.59-63
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• 2009

Calcium ion ($Ca^{2+}$) is thought to play the important role as a second messenger for signal transduction that results in various physiological responses to cope with developmental programs and environmental changes in plant. In plant cells, the central vacuole functions as a major calcium store, which is important for both signal transduction and preventing cytotoxicity. Although there is evidence for the biochemical characterizations of a calmodulin-regulated $Ca^{2+}$-ATPase (ACA11) localized to vacuole membrane, the biological function to ACA11 in plant has not been verified. In this study, we show that the cell death as the hypersensitive response (HR) in mature leaves is induced in transgenic plant of a vacuole ACA-type $Ca^{2+}$-ATPase, ACA11. Evidence that cell death phenotype is the result of ACA11 gene silencing is provided by Western blot assay using membrane fraction proteins extracted from transgenic plant. The 3, 3'-diaminobenzidine (DAB) staining study provides that the cell death is caused by the increase of reactive oxygen species (ROS) in mature leaves of transgenic plants.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.3
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• pp.1037-1047
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• 2016

HOX transcription factors are evolutionarily conserved in many different species and are involved in important cellular processes such as morphogenesis, differentiation, and proliferation. They have also recently been implicated in carcinogenesis, but their precise role in cancer, especially in cervical cancer (CC), remains unclear. In this work, using microarray assays followed by the quantitative polymerase chain reaction (qPCR), we found that the expression of 25 HOX genes was downregulated in CC derived cell lines compared with non-tumorigenic keratinocytes. In particular, the expression of HOXA9 was observed as down-modulated in CC-derived cell lines. The expression of HOXA9 has not been previously reported in CC, or in normal keratinocytes of the cervix. We found that normal CC from women without cervical lesions express HOXA9; in contrast, CC cell lines and samples of biopsies from women with CC showed significantly diminished HOXA9 expression. Furthermore, we found that methylation at the first exon of HOXA9 could play an important role in modulating the expression of this gene. Exogenous restoration of HOXA9 expression in CC cell lines decreased cell proliferation and migration, and induced an epithelial-like phenotype. Interestingly, the silencing of human papilloma virus (HPV) E6 and E7 oncogenes induced expression of HOXA9. In conclusion, controlling HOXA9 expression appears to be a necessary step during CC development. Further studies are needed to delineate the role of HOXA9 during malignant progression and to afford more insights into the relationship between downmodulation of HOXA9 and viral HPV oncoprotein expression during cercical cancer development.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.7
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• pp.3223-3228
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• 2012

HMGN5 is a typical member of the HMGN (high mobility group nucleosome-binding protein) family which may function as a nucleosomal binding and transcriptional activating protein. Overexpression of HMGN5 has been observed in several human tumors but its role in tumorigenesis has not been fully clarified. To investigate its significance for human lung cancer progression, we successfully constructed a shRNA expression lentiviral vector in which sense and antisense sequences targeting the human HMGN5 were linked with a 9-nucleotide loop. Inhibitory effects of siRNA on endogenous HMGN5 gene expression and protein synthesis were demonstrated via real-time RT-PCR and western blotting. We found HMGN5 silencing to significantly inhibit A549 and H1299 cell proliferation assessed by MTT, BrdU incorporation and colony formation assays. Furthermore, flow cytometry analysis showed that specific knockdown of HMGN5 slowed down the cell cycle at the G0/G1 phase and decreased the populations of A549 and H1299 cells at the S and G2/M phases. Taken together, these results suggest that HMGN5 is directly involved in regulation cell proliferation in A549 and H1299 cells by influencing signaling pathways involved in cell cycle progression. Thus, our finding suggests that targeting HMGN5 may be an effective strategy for human lung cancer treatment.

• Korean Journal of Head & Neck Oncology
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• v.33 no.1
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• pp.21-29
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• 2017

Methylation of CpG islands in the promoter region of genes acts as a significant mechanism of epigenetic gene silencing in head and neck squamous cell carcinoma (HNSCC). DNA methylation markers are particularly advantageous because DNA methylation is an early event in tumorigenesis, and the epigenetic modification, 5-methylcytosine, is a stable mark. In the present study, we assessed the genome-wide preliminary screening and were to identify novel methylation biomarker candidate in HNSCC. Genome-wide methylation analysis was performed on 10 HNSCC tumors using the Methylated DNA Isolation Assay (MeDIA) CpG island microarray. Validation was done using immunohistochemistry using tissue microarray of 135 independent HNSCC tumors. In addition, in vitro proliferation, migration/invasion assays, RT-PCR and immunoblotting were performed to elucidate molecular regulating mechanisms. Our preliminary validation using CpG microarray data set, immunohisto-chemistry for HNSCC tumor tissues and in vitro functional assays revealed that methylation of the Homeobox B5 (HOXB5) and H6 Family Homeobox 2 (HMX2) could be possible novel methylation biomarkers in HNSCC.

• Molecules and Cells
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• v.38 no.12
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• pp.1064-1070
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• 2015

Translocator protein 18 kDa (TSPO) is a mitochondrial outer membrane protein and is abundantly expressed in a variety of organ and tissues. To date, the functional role of TSPO on vascular endothelial cell activation has yet to be fully elucidated. In the present study, the phorbol 12-myristate 13-acetate (PMA, 250 nM), an activator of protein kinase C (PKC), was used to induce vascular endothelial activation. Adenoviral TSPO overexpression (10-100 MOI) inhibited PMA-induced vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) expression in a dose dependent manner. PMA-induced VCAM-1 expressions were inhibited by Mito-TEMPO ($0.1-0.5{\mu}m$), a specific mitochondrial antioxidants, and cyclosporin A ($1-5{\mu}m$), a mitochondrial permeability transition pore inhibitor, implying on an important role of mitochondrial reactive oxygen species (ROS) on the endothelial activation. Moreover, adenoviral TSPO overexpression inhibited mitochondrial ROS production and manganese superoxide dismutase expression. On contrasts, gene silencing of TSPO with siRNA increased PMA-induced VCAM-1 expression and mitochondrial ROS production. Midazolam ($1-50{\mu}m$), TSPO ligands, inhibited PMA-induced VCAM-1 and mitochondrial ROS production in endothelial cells. These results suggest that mitochondrial TSPO can inhibit PMA-induced endothelial inflammation via suppression of VCAM-1 and mitochondrial ROS production in endothelial cells.

• Biomolecules & Therapeutics
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• v.27 no.4
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• pp.414-422
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• 2019

There is accumulating evidence that microRNAs are emerging as pivotal regulators in the development and progression of neuropathic pain. MicroRNA-15a/16 (miR-15a/16) have been reported to play an important role in various diseases and inflammation response processes. However, whether miR-15a/16 participates in the regulation of neuroinflammation and neuropathic pain development remains unknown. In this study, we established a mouse model of neuropathic pain by chronic constriction injury (CCI) of the sciatic nerves. Our results showed that both miR-15a and miR-16 expression was significantly upregulated in the spinal cord of CCI rats. Downregulation of the expression of miR-15a and miR-16 by intrathecal injection of a specific inhibitor significantly attenuated the mechanical allodynia and thermal hyperalgesia of CCI rats. Furthermore, inhibition of miR-15a and miR-16 downregulated the expression of interleukin-$1{\beta}$ and tumor-necrosis factor-${\alpha}$ in the spinal cord of CCI rats. Bioinformatic analysis predicted that G protein-coupled receptor kinase 2 (GRK2), an important regulator in neuropathic pain and inflammation, was a potential target gene of miR-15a and miR-16. Inhibition of miR-15a and miR-16 markedly increased the expression of GRK2 while downregulating the activation of p38 mitogen-activated protein kinase and $NF-{\kappa}B$ in CCI rats. Notably, the silencing of GRK2 significantly reversed the inhibitory effects of miR-15a/16 inhibition in neuropathic pain. In conclusion, our results suggest that inhibition of miR-15a/16 expression alleviates neuropathic pain development by targeting GRK2. These findings provide novel insights into the molecular pathogenesis of neuropathic pain and suggest potential therapeutic targets for preventing neuropathic pain development.

• Biomolecules & Therapeutics
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• v.32 no.1
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• pp.84-93
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• 2024

Rosmarinic acid (RA) is a phenolic ester that protects human keratinocytes against oxidative damage induced by ultraviolet B (UVB) exposure, however, the mechanisms underlying its effects remain unclear. This study aimed to elucidate the cell signaling mechanisms that regulate the antioxidant activity of RA and confirm its cyto-protective role. To explore the signaling mechanisms, we used the human keratinocyte cell line HaCaT and SKH1 hairless mouse skin. RA enhanced glutamate-cysteine ligase catalytic subunit (GCLC) and glutathione synthetase (GSS) expression in HaCaT cells in a dose- and time-dependent manner. Moreover, RA induced nuclear factor erythroid-2-related factor 2 (NRF2) nuclear translocation and activated the signaling kinases protein kinase B (AKT) and extracellular signal-regulated kinase (ERK). Treatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, the ERK inhibitor U0126, and small interfering RNA (siRNA) gene silencing suppressed RA-enhanced GCLC, GSS, and NRF2 expression, respectively. Cell viability tests showed that RA significantly prevented UVB-induced cell viability decrease, whereas the glutathione (GSH) inhibitors buthionine sulfoximine, LY294002, and U0126 significantly reduced this effect. Moreover, RA protected against DNA damage and protein carbonylation, lipid peroxidation, and apoptosis caused by UVB-induced oxidative stress in a concentration-dependent manner in SKH1 hairless mouse skin tissues. These results suggest that RA protects against UVB-induced oxidative damage by activating AKT and ERK signaling to regulate NRF2 signaling and enhance GSH biosynthesis. Thus, RA treatment may be a promising approach to protect the skin from UVB-induced oxidative damage.

• Journal of Life Science
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• v.18 no.8
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• pp.1132-1139
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• 2008

A CpG island is a short stretch of DNA in which the frequency of the CG dinucleotide is higher than other regions. CpG islands are present in the promoters and exonic regions of approximately $30{\sim}60$% of mammalian genes so they are useful markers for genes in organisms containing 5-methylcytosine in their genomes. Recent evidence supports the notion that the hypermethylation of CpG island, by silencing tumor suppressor genes, plays a major causal role in cancer, which has been described in almost every tumor types. In this respect, CpG island search by computational methods is very helpful for cancer research and computational promoter and gene predictions. I therefore developed a window program (called CpGi) on the basis of CpG island criteria defined by D. Takai and P. A. Jones. The program 'CpGi' was implemented in Visual C++ 6.0 and can determine the locations of CpG islands using diverse parameters (%GC, Obs (CpG)/Exp (CpG), window size, step size, gap value, # of CpG, length) specified by user. The analysis result of CpGi provides a graphical map of CpG islands and G+C% plot, where more detailed information on CpG island can be obtained through pop-up window. Two human contigs, i.e. AP00524 (from chromosome 22) and NT_029490.3 (from chromosome 21), were used to compare the performance of CpGi and two other public programs for the accuracy of search results. The two other programs used in the performance comparison are Emboss-CpGPlot and CpG Island Searcher that are web-based public CpG island search programs. The comparison result showed that CpGi is on a level with or outperforms Emboss-CpGPlot and CpG Island Searcher. Having a simple and easy-to-use user interface, CpGi would be a very useful tool for genome analysis and CpG island research. To obtain a copy of CpGi for academic use only, contact corresponding author.