• 대한약학회:학술대회논문집
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• 대한약학회 2001년도 Proceedings of the Pharmaceutical Society of Korea
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• pp.48-55
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• 2001

When an elongating RNA polymerase encounters DNA damage on the template strand of a transcribed gene it can either be arrested by or be transcribed through the lesion. Lesions that arrest RNA polymerases are thought to be subject to transcription-coupled repair, whereas that damage that is bypassed can cause miscoding, resulting in mutations in the transcript (transcriptional mutagenesis). We have developed a technique using a plasmid-based luciferase reporter assay to determine the extent to which a particular type of DNA base modification is capable of causing transcriptional mutagenesis in vivo. The system uses Escherichia coli strains with different DNA repair backgrounds and is designed to detect phenotypic changes caused by transcriptional mutageneis under nongrowth conditions. In addition, this method is capable of indicating the extent to which a particular DNA repair enzyme (or pathway) suppresses the occurrence of transcriptional mutagenesis. Thus, this technique provides a tool with which the effects of various genes on non-replication-dependent pathways resulting in the generation of mutant proteins can be gauged.

• 대한의생명과학회지
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• 제12권4호
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• pp.385-391
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• 2006

Heterotrimeric GTP binding proteins (G proteins) mediate signals generated by neurotransmitters and hormones Among G proteins, Go is found in a large quantity in brain and growth cone membranes of neurons. In spite of its abundance in neurons, the role of Go is not fully understood. In our previous study, we identified promyelocytic leukemia zinc finger protein (PLZF) as an interacting partner of alpha subunit of Go ($Go{\alpha}$) and confirmed their interaction employing several biochemical assays. To date, it is reported that PLZF functioned as a cell growth suppressor and a transcription repressor. To determine effect of $Go{\alpha}$ and PLZF interaction on the cellular function of PLZF, we performed luciferase reporter gene assay and BrdU incorporation assay. Co-expression of $Go{\alpha}$ and PLZF synergistically increased the effect of PLZF alone. These results suggest that $Go{\alpha}$ may act as cellular activator of PLZF. This novel feature of Go may provide insights into understanding diverse role of Go-coupled receptor as well as its cellular actions.

• 한국잠사학회:학술대회논문집
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• 한국잠사학회 2001년도 제44회 추계 학술연구 발표회
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• pp.66-66
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• 2001

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• Tuberculosis and Respiratory Diseases
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• 제48권5호
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• pp.682-698
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• 2000

Glucocorticoid receptor (GR) functions as a suppressor of inflammation by inhibiting the expression of many cytokine genes activated by NF-${\kappa}B$. The goal of this study is to investigate the mechanism by which GR repress NF-${\kappa}B$ activation in lung epithelial cells. We used A549 and BEAS-2B lung epithelia! cell lines. Using Ig$G{\kappa}$-NF-${\kappa}B$ luciferase reporter gene construct, we found that dexamethasone significantly suppressed TNF-$\alpha$-induced NF-${\kappa}B$ activation and the overexpression of GR showed dose-dependent reduction of TNF-$\alpha$-induced NF-${\kappa}B$ activity in both cell lines. However, DNA binding of NF-${\kappa}B$ induced by TNF-$\alpha$ in electromobility shift assay was not inhibited by dexamethasone. Super shift assay with anti-p65 antibody demonstrated the existence of p65 in NF-${\kappa}B$ complex induced by $\alpha$ Western blot showed that $I{\kappa}B{\alpha}$ degradation induced by TNF-$\alpha$ was not affected by dexamethasone and $I{\kappa}B{\kappa}$ was not induced by dexamethasone, neither. To evaluate p65 specific transactivation, we adopted co-transfection study of Gal4-p65TA1 or TA2 fusion protein expression system together with 5xGal4-luciferase vector. Co-transfection of GR with Gal4-p65TA1 or TA2 repressed luciferase activity profoundly to the level of 10-20% of p65TA1- or TA2-induced transcriptional activity. And this transrepressional effect was abolished by co-transfection of CBP of SRC-1 expression vectors. These results suggest that GR-mediated transrepression of NF-${\kappa}B$ in lung epithelial cells is through competing for binding to limiting amounts of transcriptional coactivators, CBP or SRC-1.

• 생명과학회지
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• 제24권7호
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• pp.798-803
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• 2014

Cathepsin D (CtsD)는 아스파르트산 단백질 분해효소로서 cytochrome C의 방출을 유도하여 apoptosis 기전을 활성화시킨다. 본 연구에서는 3T3-L1 지방전구세포에서 CtsD 발현 조절에 관여하는 microRNA에 대해 조사하였다. 먼저 지방전구세포 사멸시 CtsD 발현 변화를 관찰하기 위하여 DNA damage agent인 doxorubicin을 3T3-L1 세포주에 노출시켜 CtsD 발현이 증가함을 확인하였다. 또한 지방전구세포주에서 CtsD가 과발현되면 세포 생존율이 감소하였다. miRanda program을 이용하여 CtsD 유전자를 표적으로 하는 microRNA를 탐색하여 miR-145를 선발하였다. Luciferase reporter assay에 의해 miR-145가 CtsD 유전자의 3' UTR 부위에 결합하여 luciferase 활성을 감소시킴을 관찰하였다. 3T3-L1 세포주에 miR-145 mimic을 도입한 결과 CtsD mRNA 발현과 단백질 수준이 감소하였다. 또한 세포주에 doxorubicin을 처리한 결과 CtsD 유전자 발현 증가와 상반되게 miR-145 발현이 감소하였다. 이외에도 miR-145 inhibitor을 세포에 도입하면 세포 생존율이 감소하였다. 이러한 결과는 지방전구세포의 세포사멸에 CtsD가 관여할 수 있으며, miR-145에 의해 CtsD 발현이 직접 조절되고 있음을 나타낸다. 따라서, 지방전구세포의 사멸을 유도하기 위해서는 miR-145 발현 제어가 주요한 표적이 될 수 있을 것으로 생각된다. 본 연구결과는 향후 비만 예방 및 치료를 위한 지방세포 사멸기전 규명에 중요한 기초 자료를 제공할 수 있을 것으로 기대한다.

• Molecules and Cells
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• 제45권7호
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• pp.465-478
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• 2022

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate the expression of target messenger RNA (mRNA) complementary to the 3' untranslated region (UTR) at the post-transcriptional level. Hsa-miR-422a, which is commonly known as miRNA derived from transposable element (MDTE), was derived from short interspersed nuclear element (SINE). Through expression analysis, hsa-miR-422a was found to be highly expressed in both the small intestine and liver of crab-eating monkey. AT-Rich Interaction Domain 5 B (ARID5B) was selected as the target gene of hsa-miR-422a, which has two binding sites in both the exon and 3'UTR of ARID5B. To identify the interaction between hsa-miR-422a and ARID5B, a dual luciferase assay was conducted in HepG2 cell line. The luciferase activity of cells treated with the hsa-miR-422a mimic was upregulated and inversely downregulated when both the hsa-miR-422a mimic and inhibitor were administered. Nuclear factor erythroid-2 (NF-E2) was selected as the core transcription factor (TF) via feed forward loop analysis. The luciferase expression was downregulated when both the hsa-miR-422a mimic and siRNA of NF-E2 were treated, compared to the treatment of the hsa-miR-422a mimic alone. The present study suggests that hsa-miR-422a derived from SINE could bind to the exon region as well as the 3'UTR of ARID5B. Additionally, hsa-miR-422a was found to share binding sites in ARID5B with several TFs, including NF-E2. The hsa-miR-422a might thus interact with TF to regulate the expression of ARID5B, as demonstrated experimentally. Altogether, hsa-miR-422a acts as a super enhancer miRNA of ARID5B by collaborating with TF and NF-E2.

• Asian Pacific Journal of Cancer Prevention
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• 제15권16호
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• pp.6949-6954
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• 2014

Background: Recent studies have indicated that microRNA-15a (miR-15a) is dysregulated in breast cancer (BC). We aimed to evaluate the expression of miR-15a in BC tissues and corresponding para-carcinoma tissues. We also focused on effects of miR-15a on cellular behavior of MDA-MB-231 and expression of its target gene synuclein-${\gamma}$ (SNCG). Materials and Methods: The expression levels of miR-15a were analysed in BC formalin fixed paraffin embedded (FFPE) tissues by microarray and quantitative real-time PCR. CCK-8 assays, cell cycle and apoptosis assays were used to explore the potential functions of miR-15a in MDA-MB-231 human BC cells. A luciferase reporter assay confirmed direct targets. Results: Downregulation of miR-15a was detected in most primary BCs. Ectopic expression of miR-15a promoted proliferation and suppressed apoptosis in vivo. Further studies indicated that miR-15a may directly interact with the 3'-untranslated region (3'-UTR) of SNCG mRNA, downregulating its mRNA and protein expression levels. SNCG expression was negatively correlated with miR-15a expression. Conclusions: MiR-15a has a critical role in mediating cell cycle arrest and promoting cell apoptosis of BC, probably by directly targeting SNCG. Thus, it may be involved in development and progression of BC.

• 동의신경정신과학회지
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• 제20권1호
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• pp.147-164
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• 2009

Objectives : We investigated the effects of Sopungsungi-won(Shu!engshunqiyuan) (SSEx1, SSEx2) to treat the metabolic syndrome by the molecular mechanism of regulation of PPAR and modulation of mitochondrial MCAD, VLCAD mRNA expression. Methods : Mouse NMu2Li liver cells and C2C12 skeletal muscle myogenic progenital cells were transiently transfected with expression plasmids for PPAR(PPAR${\alpha}$, PPAR${\delta}$), a luciferase reporter gene construct containing 3 copies of the PPRE from the rat acyl-CoA oxidase gene and ${\beta}$-galactosidase gene. Cells were treated with several concentrated kinds of SSEx1, SSEx2 at the initial time of culture and analyzed PPAR${\alpha}$, PPAR${\delta}$ reporter gene activity using spectrophotometer (405 nm). Total RNA was extracted from SSEx1, SSEx2 and measured mRNA levels of mitochondrial MCAD, VLCAD. Representative RT-PCR bands are shown. Results : 1. SSEx1 increased the expression of PPAR${\alpha}$ reporter gene activities at 0.1 ${\mu}$g/ml (p${\mu}$g/ml (p<0.05), SSEx2 at 0.1 ${\mu}$g/ml (p${\mu}$g/ml (p<0.05) significantly in NMu2Li liver cell lines. 2. SSEx1 increased the expression of PPAR${\alpha}$ reporter gene activities at 1 ${\mu}$g/ml (p${\mu}$g/ml (p${\alpha}$ reporter gene activities in C2C12 skeletal muscle cells. 4. SSEx1 increased the modulation of mitochondrial MCAD mRNA expression (p<0.05) significantly in NMu2Li liver cell lines. 5. SSEx1, SSEx2 both increased the modulation of mitochondrial MCAD mRNA expression (p<0.05) significantly in C2C12 skeletal muscle cells. Conclusions : These results show the SSEx1, SSEx2 can be used as therapeutic agent for metabolic syndrome and it's molecular mechanisms of PPAR more contribute to the activation of PPAR${\alpha}$ then PPAR${\delta}$ reporter gene activities and it's total RNA more contribute to the modulation of mitochondrial MCAD then VLCAD mRNA expression.

• 동의생리병리학회지
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• 제21권1호
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• pp.243-249
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• 2007

The French paradox has been attributed to the antioxidant properties of flavonoids present in the red wine. Amentoflavone(AF) is a bi-flavonoid compound with anti-fungal and anti-inflammatory activities. We isolated AF from Selaginella tamariscina, and studied its effects on nuclear factor-B(NF-B)-mediated MMP-9 gene expression in RAW264.7 cells. AF blocked the lipopolysaccharide(LPS)-induced expression of MMP-9. Zymographic and immunoblot analyses showed that AF suppressed LPS-induced MMP-9 expression in a dose-dependent manner. To clarify the mechanistic basis for its inhibition of MMP-9 induction, we examined the effect of AF on the transactivation of MMP-9 gene by luciferase reporter activity using -1.59 kb flanking region. AF potently suppressed the reporter gene activity. This inhibition was characterized by down-regulation of MMP-9, which was transcriptionally regulated at NF-B site and activation protein-1 (AP-1) site in the MMP-9 promoter, two important nuclear transcription factors that are involved in MMP-9 expression. These findings indicate the efficacy of AF in inhibiting MMP-9 expression through the transcription factors NF-B and AP-1 on LPS-induced RAW264.7 cells.

• Preventive Nutrition and Food Science
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• 제21권1호
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• pp.62-67
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• 2016

Green tea (Camellia sinensis) is one of the most popular beverages in the world and has been acknowledged for centuries as having significant health benefits. (-)-Epigallocatechin-3-gallate (EGCG) is the most abundant catechin in green tea, and it has been reported to have health benefit effects. Peroxisome proliferator-activated receptor ${\gamma}$ coactivator $(PGC)-1{\alpha}$ is a crucial regulator of mitochondrial biogenesis and hepatic gluconeogenesis. The objective of this study was to investigate whether EGCG from green tea can affect the ability of transcriptional regulation on $PGC-1{\alpha}$ mRNA expression in HepG2 cells and 3T3-L1 adipocytes. To study the molecular mechanism that allows EGCG to control $PGC-1{\alpha}$ expression, the promoter activity levels of $PGC-1{\alpha}$ were examined. The $PGC-1{\alpha}$ mRNA level was measured using quantitative real-time PCR. The -970/+412 bp of $PGC-1{\alpha}$ promoter was subcloned into the pGL3-Basic vector that includes luciferase as a reporter gene. EGCG was found to up-regulate the $PGC-1{\alpha}$ mRNA levels significantly with $10{\mu}mol/L$ of EGCG in HepG2 cells and differentiated 3T3-L1 adipocytes. $PGC-1{\alpha}$ promoter activity was also increased by treatment with $10{\mu}mol/L$ of EGCG in both cells. These results suggest that EGCG may induce $PGC-1{\alpha}$ gene expression, potentially through promoter activation.