• Biomedical Science Letters
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• v.23 no.3
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• pp.272-276
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• 2017

HOX genes are transcription factors that play important roles in body patterning and cell fate specification during normal development. In previous study, we found aberrant overexpression of HOXB5 in breast cancer tissues and cell lines, and demonstrated that HOXB5 is important in regulation of cell proliferation, tamoxifen resistance, and invasiveness through the epithelial-mesenchymal transition (EMT). Although the relationship between HOXB5 and phenotypic changes in MCF7 breast cancer cells has been studied, the molecular function of HOXB5 as a transcription factor remains unclear. IL-6 has been reported to be involved in not only inflammation but also cancer progression, which is characterized by the increase of growth speed and invasiveness of tumor cells. In this study, we selected Interleukin-6 (IL-6) as HOXB5 putative downstream target gene and discovered that HOXB5 transcriptionally up-regulated the expression of IL-6 in HOXB5 overexpressing MCF7 cells. The upstream region (~1.2 kb) of IL-6 promoter turned out to contain several putative HOX consensus binding sites. Chromatin immunoprecipitation assay confirmed that HOXB5 directly binds to the promoter region of IL-6 and positively regulated the expression of IL-6. These data all together, indicate that HOXB5 promotes IL-6 transcription by actively binding to the putative binding sites located in the upstream region of IL-6, which enable to increase its promoter activity in MCF7 breast cancer cells.

• BMB Reports
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• v.50 no.9
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• pp.472-477
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• 2017

The transcription repressor Bach2 has been proposed as a regulator of T cell quiescence, but the underlying mechanism is not fully understood. Given the importance of interleukin-2 in T cell activation, we investigated whether Bach2 is a component of the network of factors that regulates interleukin-2 expression. In primary and transformed $CD4^+$ T cells, Bach2 overexpression counteracted T cell receptor/CD28- or PMA/ionomycin-driven induction of interleukin-2 expression, and silencing of Bach2 had the opposite effect. Luciferase and chromatin immunoprecipitation assays revealed that Bach2 binds to multiple Maf-recognition element-like sites on the interleukin-2 proximal promoter in a manner competitive with AP-1, and thereby represses AP-1-driven induction of interleukin-2 transcription. Thus, this study demonstrates that Bach2 is a direct repressor of the interleukin-2 gene in $CD4^+$ T cells during the immediate early phase of AP-driven activation, thereby playing an important role in the maintenance of immune quiescence in the steady state.

• The Journal of Korean Society of Virology
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• v.28 no.1
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• pp.53-62
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• 1998

Human papillomavirus (HPV) 16, E7 proteins derived from the prototype (Bac73) and natural variant (Bac101) E7 open reading frame were produced in Sf9 insect cells. The variant E7 gene occurred naturally by substitution mutation at the position of 88 nucleotide, resulting serine instead of asparagine. Using E7 specific monoclonal antibody (VD6), both E7 proteins were identified in recombinant baculovirus infected SF9 cells. Radiolabelling and immunoprecipitation analysis revealed that both E7 proteins were phosphoproteins. Immunostaining result showed that E7 proteins were mainly localized in the cytoplasm. Nuclear form of E7 proteins was also detected after a sequential fractionation procedure for removing chromatin structure. Considering that the VD6 recognition site in E7 protein is located within 10 amino acid at the N-terminus, this region appears to be blocked by the nuclear component. Western blot analysis revealed that nuclear form was more abundant than cytoplasmic E7 proteins. Time course immunostaining showed that the primary location of E7 protein was the nucleus and exported to the cytoplasm as proteins were accumulated. These events occurred similarly in both Bac73 and Bac101 infected Sf9 cells, suggesting that these two proteins may have similar biological functions.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.10
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• pp.1674-1682
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• 2020

Objective: This study aimed to elucidate the effect of miR-140 on the proliferation of porcine fetal fibroblasts (PFFs) and identify the target genes of miR-140 in PFFs. Methods: In this study, bioinformatics software was used to predict and verify target genes of miR-140. Quantitative polymerase chain reaction and western blot were used to detect the relationship between miR-140 and its target genes in PFFs. Dual luciferase reporter gene assays were performed to assess the interactions among miR-140, type 1 insulin-like growth factor receptor (IGF1R), and SRY-box 4 (SOX4). The effect of miR-140 on the proliferation of PFFs was measured by CCK-8 when PFFs were transfected with a miR-140 mimic or inhibitor. The transcription factor SOX4 binding to promoter of IGF1R was detected by chromatin immunoprecipitation assay (ChIP). Results: miR-140 directly targeted IGF1R and inhibited proliferation of PFFs. Meanwhile, miR-140 targeted transcription factor SOX4 that binds to promoter of porcine IGF1R to indirectly inhibit the expression of IGF1R. In addition, miR-140 inhibitor promoted PFFs proliferation, which is abrogated by SOX4 or IGF1R knockdown. Conclusion: miR-140 inhibited PFFs proliferation by directly targeting IGF1R and indirectly inhibiting IGF1R expression via SOX4, which play an important role in the development of porcine fetal.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.13
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• pp.5181-5185
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• 2015

Background: CCAT1 has been reported to be linked with pathogenesis of malignancies including colon cancer and gastric cancer. However, the regulatory effect of CCAT1 in hepatocellular carcinoma (HCC) remains unclear. The purpose of this research was to identify any role of CCAT1 in the progression of HCC. Materials and Methods: Real time-PCR was performed to test the relative expression of CCAT1 in HCC tissues. A computation screen of CCAT1 promoter was conducted to search for transcription-factor-binding sites. The association of c-Myc with CCAT1 promoter in vivo was tested by Pearson correlation analysis and chromatin immunoprecipitation assay. Additionally, Kaplan-Meier analysis and Cox proportional hazards analyses were performed. Results: c-Myc directly binds to the E-box element in the promoter region of CCAT, and when ectopically expressed increases promoter activity and expression of CCAT1. Moreover, Kaplan-Meier analysis demonstrated that the patients with low expression of CCAT1 demonstrated better overall and relapse-free survival compared with the high expression group. Cox proportional hazards analyses showed that CCAT1 expression was an independent prognostic factor for HCC patients. Conclusions: The findings demonstrated CCAT1, acting as a potential biomarker in predicting the prognosis of HCC, is regulated by c-Myc.

• International Journal of Oral Biology
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• v.38 no.3
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• pp.121-126
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• 2013

Tumor necrosis factor alpha ($TNF{\alpha}$) is a multifunctional inflammatory cytokine that regulates various cellular and biological processes. Increased levels of $TNF{\alpha}$ have been implicated in a number of human diseases including diabetes and arthritis. Sympathetic nervous system stimulation via the beta2-adrenergic receptor (${\beta}2AR$) in osteoblasts suppresses osteogenic activity. We previously reported that $TNF{\alpha}$ upregulates ${\beta}2AR$ expression in murine osteoblastic cells and that this modulation is associated with $TNF{\alpha}$ inhibition of osteoblast differentiation. In our present study, we explored whether $TNF{\alpha}$ induces ${\beta}2AR$ expression in human osteoblasts and then identified the downstream signaling pathway. Our results indicated that ${\beta}2AR$ expression was increased in Saos-2 and C2C12 cells by $TNF{\alpha}$ treatment, and that this increase was blocked by the inhibition of NF-${\kappa}B$ activation. Chromatin immunoprecipitation and luciferase reporter assay results indicated that NF-${\kappa}B$ directly binds to its cognate elements on the ${\beta}2AR$ promoter and thereby stimulates ${\beta}2AR$ expression. These findings suggest that the activation of $TNF{\alpha}$ signaling in osteoblastic cells leads to an upregulation of ${\beta}2AR$ and also that $TNF{\alpha}$ induces ${\beta}2AR$ expression in an NF-${\kappa}B$-dependent manner.

• BMB Reports
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• v.40 no.1
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• pp.88-94
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• 2007

Matrix metalloproteinase-9 (MMP-9) plays a pivotal role in the turnover of extracellular matrix (ECM) and in the migration of normal and tumor cells in response to normal physiologic and numerous pathologic conditions. Here, we show that the transcription of the MMP-9 gene is induced by lipopolysaccharide (LPS) stimulation in cells of a macrophage lineage (RAW 264.7 cells). We provide evidence that the NF-$\kappa$B binding site of the MMP-9 gene contributes to its expression in the LPS-signaling pathway, since mutation of NF-$\kappa$B binding site of MMP-9 promoter leads to a dramatic reduction in MMP-9 promoter activation. In addition, the degradation of l$\kappa$B$\alpha$;, and the presences of myeloid differentiation protein (MyD88) and tumor necrosis factor receptor-associated kinase 6 (TRAF6) were found to be required for LPS-activated MMP-9 expression. Chromatin immunoprecipitation (ChIP) assays showed that functional interaction between NF-$\kappa$B and the MMP-9 promoter element is necessary for LPS-activated MMP-9 induction in RAW 264.7 cells. In conclusion, our observations demonstrate that NF-$\kappa$B contributes to LPS-induced MMP-9 gene expression in a mouse macrophage cell line.

• Molecules and Cells
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• v.40 no.1
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• pp.54-65
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• 2017

Although cancer/testis antigen DDX53 confers anti-cancer drug-resistance, the effect of DDX53 on cancer stem cell-like properties and autophagy remains unknown. MDA-MB-231 ($CD133^+$) cells showed higher expression of DDX53, SOX-2, NANOG and MDR1 than MDA-MB-231 ($CD133^-$). DDX53 increased in vitro self-renewal activity of MCF-7 while decreasing expression of DDX53 by siRNA lowered in vitro self-renewal activity of MDA-MB-231. DDX53 showed an interaction with EGFR and binding to the promoter sequences of EGFR. DDX53 induced resistance to anti-cancer drugs in MCF-7 cells while decreased expression of DDX53 by siRNA increased the sensitivity of MDA-MB-231 to anti-cancer drugs. Negative regulators of DDX53, such as miR-200b and miR-217, increased the sensitivity of MDA-MB-231 to anti-cancer drugs. MDA-MB-231 showed higher expression of autophagy marker proteins such as ATG-5, $pBeclin1^{Ser15}$ and LC-3I/II compared with MCF-7. DDX53 regulated the expression of marker proteins of autophagy in MCF-7 and MDA-MB-231 cells. miR-200b and miR-217 negatively regulated the expression of autophagy marker proteins. Chromatin immunoprecipitation assays showed the direct regulation of ATG-5. The decreased expression of ATG-5 by siRNA increased the sensitivity to anti-cancer drugs in MDA-MB-231 cells. In conclusion, DDX53 promotes stem cell-like properties, autophagy, and confers resistance to anti-cancer drugs in breast cancer cells.

• Korean Journal of Microbiology
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• v.53 no.1
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• pp.49-54
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• 2017

Sus1 / ENY2 is a tiny conserved protein that is involved in chromatin remodeling and mRNA biogenesis. Sus1 is associated to two nuclear complexes, the transcriptional coactivator SAGA and the nuclear pore associated TREX2. In fission yeast, Schizosaccharomyces pombe, ortholog of Sus1 / ENY2 was identified from the genome database. Tetrad analysis showed that the S. pombe sus1 is not essential for growth. However, deletion of the sus1 gene caused cold-sensitive growth retardation with slight accumulation of $poly(A)^+$ RNA in the nucleus. And the Sus1-GFP protein is localized mainly in the nucleus. Yeast two-hybrid analysis and co-immunoprecipitation experiment showed that Sus1 interacts with Sac3, another subunit of TREX2 complex. These results suggest that S. pombe Sus1 is also involved in mRNA export from the nucleus as a component of TREX-2 complex.

• Molecules and Cells
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• v.26 no.4
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• pp.409-414
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• 2008

The cholesteryl ester transfer protein (CETP), a key player in cholesterol metabolism, has been shown to promote the transfer of triglycerides from very low density lipoprotein (VLDL) and low density lipoprotein (LDL) to high density lipoprotein (HDL) in exchange for cholesterol ester. Here we demonstrate that farnesoid X receptor ${\alpha}$ ($FXR{\alpha}$; NR1H4) down-regulates CETP expression in HepG2 cells. A $FXR{\alpha}$ ligand, chenodeoxycholic acid (CDCA), suppressed basal mRNA levels of the CETP gene in HepG2 cells in a dose-dependent manner. Using gel shift and chromatin immunoprecipitation (ChIP) assays, we found that $FXR{\alpha}$ could bind to the liver X receptor ${\alpha}$ ( $LXR{\alpha}$; NR1H3) binding site (LXRE; DR4RE) located within the CETP 5' promoter region. $FXR{\alpha}$ suppressed $LXR{\alpha}$-induced DR4RE-luciferase activity and this effect was mediated by a binding competition between $FXR{\alpha}$ and $LXR{\alpha}$ for DR4RE. Furthermore, the addition of CDCA together with a $LXR{\alpha}$ ligand, GW3965, to HepG2 cells was shown to substantially decrease mRNA levels of hepatic CETP gene, which is typically induced by GW3965. Together, our data demonstrate that $FXR{\alpha}$ down-regulates CETP gene expression via binding to the DR4RE sequence within the CETP 5' promoter and this $FXR{\alpha}$ binding is essential for $FXR{\alpha}$ inhibition of $LXR{\alpha}$-induced CETP expression.