• Journal of Life Science
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• v.20 no.2
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• pp.275-280
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• 2010

Sulforaphane is a naturally occurring member of the iosothiocyanate family, which reveals chemopreventive capacities including anti-cancer, anti-inflammation and inhibition of MMP-9 activities. In this study, we investigated the effect of sulforaphane on the expression of matrix metalloproteinase-9 (MMP-9) in lipopolysaccharide (LPS)-induced Raw 264.7 cells. Sulforaphane strikingly suppressed the LPS-induced MMP-9 activity and mRNA expression in a dose-dependent manner. In addition, sulforaphane inhibited not only the LPS-induced MMP-9 promoter activity but also LPS-mediated activator protein-1 (AP-1) and nuclear factor-kB (NF-${\kappa}B$) promoter activity. Transient transfection by MMP-9 constructs, in which specific transcriptional factors were mutagenized, indicated that the effects of LPS and sulforaphane were mediated via AP-1 and NF-${\kappa}B$ response elements. We found that sulforaphane had the ability to suppress LPS-induced invasion in vitro. Taken together, these results demonstrated that sulforaphane effectively suppressed LPS-induced MMP-9 expression via modulation of promoter elements (AP-1 and NF-${\kappa}B$) in MMP-9 transcriptional activation.

• Journal of Life Science
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• v.28 no.1
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• pp.37-42
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• 2018

Non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) is a transforming growth factor beta (TGF-${\beta}$) superfamily gene associated with pro-apoptotic and anti-tumorigenic activities. In the present study, we investigated if caffeic acid phenethyl ester (CAPE) derived from propolis could induce the expression of anti-tumorigenic gene NAG-1. Our results indicate that CAPE significantly induced NAG-1 expression in a time- and concentration-dependent manner in HCT116 cells. We also found that CAPE induced NAG-1 expression in a concentration-dependent manner in another human colorectal cancer cell line, LOVO. In addition, CAPE triggered apoptosis, which was detected with Western blot analysis using poly-(ADP-ribose) polymerase antibody. NAG-1 induction by CAPE was not dependent on transcription factor p53, which was confirmed with Western blot analysis using p53 null HCT116 cells. The luciferase assay results indicated that the new cis-elements candidates were located between -474 and -1,086 of the NAG-1 gene promoter. CAPE dramatically induced activating transcription factor 3 (ATF3) expression, but not cAMP response element-binding protein (CREB), which shares the same binding sites with ATF3. The co-transfection experiment with pCG-ATF3 and pCREB showed that only ATF3 was associated with NAG-1 up-regulation by CAPE, whereas CREB had no effect. In conclusion, the results suggest that CAPE could induce the expression of anti-tumorigenic gene NAG-1 mainly through ATF3.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.4
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• pp.547-555
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• 2020

Objective: Apoptosis of ovarian granulosa cells (GCs) affects mammalian follicular development and fecundity. This study aimed to explore the regulatory relationship between microRNA-26a (miR-26a) and the 3β-hydroxysteroid-Δ24-reductase gene (DHCR24) gene in porcine follicular granular cells (pGCs), and to provide empirical data for the development of methods to improve the reproductive capacity of pigs. Methods: The pGCs were transfected with miR-26a mimic, miR-26a inhibitor and DHCR24-siRNA in vitro. The cell apoptosis rate of pGCs was detected by the flow cytometry. The secretion levels of estradiol (E2) and progesterone (P) in pGCs were detected by enzyme-linked immunosorbent assay. Double luciferase validation system was used to detect the binding sites between miR-26a and DHCR24 3'-UTR region. Qualitative real-time polymerase chain reaction and Western blotting were used to verify the DHCR24 mRNA and protein expression in pGCs, respectively, after transfecting with miR-26a mimic and miR-26a inhibitor. Results: Results showed that enhancement of miR-26a promoted apoptosis, and inhibited E2 and P secretion in pGCs. Meanwhile, inhibition of DHCR24 also upregulated the Caspase-3 expression, reduced the BCL-2 expression, promoted pGCs apoptosis, and inhibited E2 and P secretion in pGCs. There were the binding sites of miR-26a located within DHCR24 3'-UTR. Up-regulation of miR-26a inhibited DHCR24 mRNA and protein expression in pGCs. Conclusion: This study demonstrates that miR-26a can promote cell apoptosis and inhibit E2 and P secretion by inhibiting the expression of DHCR24 in pGCs.

• Biomolecules & Therapeutics
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• v.17 no.1
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• pp.70-78
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• 2009

Coptis japonica (C. japonica) is a perennial medicinal plant that has anti-inflammatory activity. C. japonica contains numerous biologically active alkaloids including berberine, palmatine, epi-berberine, and coptisine. The most well-known anti-inflammatory principal in C. japonica is berberine. For example, berberine has been implicated in the inhibition of iNOS induction by cytokines in microglial cells. However, the efficacies of other alkaloids components on microglial activation were not investigated yet. In this study, we investigated the effects of three alkaloids (palmatine, epi-berberine and coptisine) from C. japonica on lipopolysaccharide (LPS)-induced microglial activation. BV2 microglial cells were immunostimulated with LPS and then the production of several inflammatory mediators such as nitric oxide (NO), reactive oxygen species (ROS) and matrix metalloproteinase-9 (MMP-9) were examined as well as the phosphorylation status of Erk1/2 mitogen activated protein kinase (MAPK). Palmatine and to a lesser extent epi-berberine and coptisine, significantly reduced the release of NO, which was mediated by the inhibition of LPS-stimulated mRNA and protein induction of inducible nitric oxide synthase (iNOS) from BV2 microglia. In addition to NO, palmatine inhibited MMP-9 enzymatic activity and mRNA induction by LPS. Palmatine also inhibited the increase in the LPS-induced MMP-9 promoter activity determined by MMP-9 promoter luciferase reporter assay. LPS stimulation increased Erk1/2 phosphorylation in BV2 cells and these alkaloids inhibited the LPS-induced phosphorylation of Erk1/2. The anti-inflammatory effect of palmatine in LPS-stimulated microglia may suggest the potential use of the alkaloids in the modulation of neuroinflammatory responses, which might be important in the pathophysiological events of several neurological diseases including Alzheimer's disease (AD), multiple sclerosis (MS), Parkinson's disease (PD) and stroke.

• Journal of Life Science
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• v.24 no.1
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• pp.1-7
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• 2014

Eukaryotic gene expression is an important process, which is initiated by several transcription factors and RNA polymerases that occupy the promoter region of genomic DNA. Although there are many experiments to identify the promoter region in a gene, it is time and labor consuming to finalize it. In this study, we utilized bioinformatic programs, including Ensembl, NCBI, and CpG plots, to identify the cloning promoter region in SETDB1 genomic DNA. We performed PCR amplification to obtain the SETDB1 promoter on an approximately 2 kb region upstream from the TSS named SETDB1-P1. The PCR product was ligated with TA cloning vectors, and we confirmed the insert size using restriction enzyme digestion. Sequentially, the insert was subcloned into a pGL3-luc vector to produce pGL3-SETDB1- P1-luc and then confirmed by DNA sequencing. We also obtained a fragmented PCR product called P2 and P3 and performed a luciferase assay using pGL3-SETDB1-P1-luc transfection. We found that several anticancer drugs, including taxol, 4-FU, and doxorubicin, decreased the promoter activity of SETDB1. We obtained consistent data on the regulation of SETDB1 gene expression after anticancer drug treatment using Western blot analysis and RT-PCR. Our results suggest that promoter cloning of the human SETDB1 gene utilizing bioinformatics is a very useful and timesaving approach to study gene expression.

• Journal of Life Science
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• v.24 no.7
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• pp.777-783
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• 2014

Cathepsin D (CtsD), an aspartyl peptidase, is involved in apoptosis, resulting in the release of cytochrome C from mitochondria in cells. Here, we investigated microRNA regulation of CtsD expression in 3T3-L1 cells. First, we observed the expression of CtsD in cells in response to doxorubicin (Dox). As expected, the level of CtsD mRNA increased in 3T3-L1 cells exposed to Dox in a dose-dependent manner. The cellular viability of ectopically expressed CtsD cells was decreased. Next, we used the miRanda program to search for particular microRNA targeting CtsD. MiR-145 was selected as a putative controller of CtsD because it had a high mirSVR score. In a reporter assay, the luciferase activity of cells containing the CtsD 3'-UTR region decreased in cells transfected with a miR-145 mimic compared to that of a control. The level of CtsD expression was down-regulated in preadipocytes ectopically expressing miR-145 and up-regulated by an miR-145 inhibitor. Cells also suppressed miR-145 expression when exposed to Dox. The miR-145 inhibitor reduced the cellular viability of 3T3-L1 cells. Taken together, these data suggest that miR-145 regulates CtsD-mediated cell death in adipocytes. These findings may have valuable implications concerning the molecular mechanism of CtsD-mediated cell death in obesity, suggesting that CtsD could be a useful therapeutic tool for the prevention and treatment of obesity by regulating fat cell numbers.

• Journal of Ginseng Research
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• v.40 no.4
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• pp.423-430
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• 2016

Background: The induction of cellular defensive genes such as phase II detoxifying and antioxidant enzymes is a highly effective strategy for protection against carcinogenesis as well as slowing cancer development. Transcription factor Nrf2 (nuclear factor E2-related factor 2) is responsible for activation of phase II enzymes induced by natural chemopreventive compounds. Methods: Red ginseng oil (RGO) was extracted using a supercritical $CO_2$ extraction system and chemical profile of RGO was investigated by GC/MS. Effects of RGO on regulation of the Nrf2/antioxidant response element (ARE) pathway were determined by ARE-luciferase assay, western blotting, and confocal microscopy. Results: The predominant components of RGO were 9,12-octadecadienoic acid (31.48%), bicyclo[10.1.0] tridec-1-ene (22.54%), and 22,23-dihydrostigmasterol (16.90%). RGO treatment significantly increased nuclear translocation of Nrf2 as well as ARE reporter gene activity, leading to upregulation of heme oxygenase-1 and NAD(P)H:quinone oxidoreductase 1. Phosphorylation of the upstream kinases such as apoptosis signal-regulating kinase (ASK)1, mitogen-activated protein kinase (MAPK) kinase (MKK)4/7, c-Jun N-terminal kinase (JNK), and p38 MAPK were enhanced by treatment with RGO. In addition, RGO-mediated Nrf2 expression and nuclear translocation was attenuated by JNK inhibitor SP600125 and p38 MAPK inhibitor SB202190. Conclusion: RGO could be used as a potential chemopreventive agent, possibly by induction of Nrf2/ARE-mediated phase II enzymes via ASK1-MKK4/7-JNK and p38 MAPK signaling pathways.

• Journal of Life Science
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• v.17 no.7 s.87
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• pp.923-930
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• 2007

Thymus can regenerate to its normal mass within 14 days after acute involution induced by cyclophosphamide (CY) in adult rat. Despite the established role of Wnt pathways in the process of thymus development, they have not yet been associated with the regeneration of adult thymus. The purpose of this study was to investigate whether Wnt7b, which is expressed in developing thymic epithelial cells rather than in thymocytes, is modulated during thymic regeneration in adult rat. Here, we show that Wnt7b expression was up-regulated in the regenerating thymus. Cells immunolabeled for the Wnt7b were identified as macrophages. Furthermore, Wnt7b gene expression was decreased by the treatment of receptor activator of NF-kappaB ligand (RANKL). Taken together, our results demonstrate that Wnt7b gene expression was increased in macrophages during thymic regeneration and negatively regulated by RANKL.

• Molecules and Cells
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• v.41 no.9
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• pp.830-841
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• 2018

Recent studies have indicated that microRNAs (miRNAs) play an important role in hepatocellular carcinoma (HCC) progression. In this study, we showed that miR-766-3p was decreased in approximately 72% of HCC tissues and cell lines, and its low expression level was significantly correlated with tumour size, TNM stage, metastasis, and poor prognosis in HCC. Ectopic miR-766-3p expression inhibited HCC cell proliferation, colony formation, migration and invasion. In addition, we showed that miR-766-3p repressed Wnt3a expression. A luciferase reporter assay revealed that Wnt3a was a direct target of miR-766-3p, and an inverse correlation between miR-766-3p and Wnt3a expression was observed. Moreover, Wnt3a up-regulation reversed the effects of miR766-3p on HCC progression. In addition, our study showed that miR-766-3p up-regulation decreased the nuclear ${\beta}-catenin$ level and expression of Wnt targets (TCF1 and Survivin) and reduced the level of MAP protein regulator of cytokinesis 1 (PRC1). However, these effects of miR-766-3p were reversed by Wnt3a up-regulation. In addition, PRC1 upregulation increased the nuclear ${\beta}-catenin$ level and protein expression of TCF1 and Survivin. iCRT3, which disrupts the ${\beta}-catenin-TCF4$ interaction, repressed the TCF1, Survivin and PRC1 protein levels. Taken together, our results suggest that miR-766-3p down-regulation promotes HCC cell progression, probably by targeting the Wnt3a/PRC1 pathway, and miR-766-3p may serve as a potential therapeutic target in HCC.

• International Journal of Oral Biology
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• v.32 no.4
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• pp.119-125
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• 2007

Dlx3 is a homeodomain protein and is known to play a role in development and differentiation of many tissues. Deletion of four base pairs in DLX3 (NT3198) is causally related to tricho-dento-osseous (TDO) syndrome (OMIM #190320), a genetic disorder manifested by taurodontism, hair abnormalities, and increased bone density in the cranium. The molecular mechanisms that explain the phenotypic characteristics of TDO syndrome have not been clearly determined. In this study, we examined phenotypic characteristics of wild type DLX3(wtDlx3) and 4-BP DEL DLX3 (TDO mtDlx3) in C2C12 cells. To investigate how wtDlx3 and TDO mtDlx3 differentially regulate osteoblastic differentiation, reporter assays were performed by using luciferase reporters containing the promoters of alkaline phosphatase, bone sialoprotein or osteocalcin. Both wtDlx3 and TDO mtDlx3 enhanced significantly all the reporter activities but the effect of mtDlx3 was much weaker than that of wtDlx3. In spite of these differences in reporter activity, electrophoretic mobility shift assay showed that both wtDlx3 and TDO mtDlx3 formed similar amounts of DNA binding complexes with Dlx3 binding consensus sequence or with ALP promoter oligonucleotide bearing the Dlx3 binding core sequence. TDO mtDlx3 exhibits a longer half-life than wtDlx3 and it corresponds to PESTfind analysis result showing that potential PEST sequence was missed in carboxy terminal of TDO mtDlx3. In addition, co-immunoprecipitation demonstrated that TDO mtDlx3 binds to Msx2 more strongly than wtDlx3. Taken together, though TDO mtDlx3 acted as a weaker transcriptional activator than wtDlx3 in osteoblastic cells, there is possibility that during in vivo osteoblast differentiation TDO mtDlx3 may antagonize transcriptional repressor activity of Msx2 more effectively and for longer period than wtDlx3, resulting in enhancement of osteoblast differentiation.