• Asian Pacific Journal of Cancer Prevention
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• v.16 no.17
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• pp.7749-7754
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• 2015

Background: Currently, cationic liposome has become the commonly used vehicles for gene transfection. Furthermore, one of the most significant steps in microRNAs expression studies is transferring microRNAs into cell cultures successfully. In this study we aim to approach the feasibility of transfection of cervical cancer cell lines mediated by liposome and to obtain the optimized transfection condition for cervical cancer cell lines. Materials and Methods: $Lipofectamine^{TM}2000$ as the carrier, miR-101 mimic was transfected into Hela cells and Siha cells. Using green fluorescent protein as reporter gene, to set different groups according to cell seeding density, the amount of miRNA, miRNA and the proportion of Liposomes, Whether to add serum into medium to study their impact on the liposomal transfection efficiency. Finally, MTT assay was used to analyze the relative minimal cell toxicity of liposome reagents. Results: The seeding density of Hela cell line and Siha are $1.5{\times}10^4$ (per well of 24 well plates), miRNA amount is 1ul of both, the ratio of miRNA and liposome is 1:0.5 of Hela cell line; 1:0.7 of Siha cell line respectively, after 24 hours we can get the highest transfection efficiency. Compared with serum medium, only Siha cells cultured with serum-free medium obtained higher transfection efficiency before transfection (P<0.01). MTT assay showed that according to the above conditions which has the lowest cytotoxicity. Conclusions: The method of Liposome to transfected is a suitable way and it can be an efficient reagent for miRNA delivery for Hela cells and Siha cells in vitro. It may serve as a reference for the further research or application.

• 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.

• BMB Reports
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• v.43 no.7
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• pp.468-473
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• 2010

Previously, various inhibitors of cell wall synthesis induced the drp35 gene of Staphylococcus aureus efficiently. To determine whether drp35 could be exploited in antistaphylococcal drug discovery, we cloned the promoter of drp35 ($P_d$) and developed different biological assay systems using an engineered S. aureus strain that harbors a chromosomally-integrated $P_d$ - lacZ transcriptional fusion. An agarose-based assay showed that $P_d$ is induced not only by the cell wall-affecting antibiotics but also by rifampicin and ciprofloxacin. In contrast, a liquid medium-based assay revealed the induction of $P_d$ specifically by the cell wall-affecting antibiotics. Induction of $P_d$ by sublethal concentrations of cell wall-affecting antibiotics was even assessable in a microtiter plate assay format, indicating that this assay system could be potentially used for high-throughput screening of new cell wall-inhibiting compounds.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1997.04a
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• pp.81-81
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• 1997

We previously demonstrated an enhancer-like positive regulatory element within a 259-bp sequence (-2352 to-2094 bp) of the human CYP1A2 gene in HepG2 cells. Three protein binding sites were identified by DNase I footprint analyses within the 259-bp sequence: protected region A PRA ( -2283 to-2243 bp), PRB (-2218 to-2187 bp), and PRC (-2124 to-2098 bp) (I. Chung and E. Bresnick, Mol. Pharmacol. 47, 677-685, 1995). In the present study, the functional significance of those protected regions was examined. Transfection experiments with deletion and substitution mutants defined the PRB and PRC as containing positive and negative regulatory elements, respectively. Human breast carcinoma MCF-7 cells were cotransfected with a hepatocyte nuclear factor-1 (HNF-1) expression vector and CYP1A2 promoter-or thymidine kinase promoter-luciferase remoter gene constructs. HNF-1, which contributes to the liver specificity of genes, enhanced reporter gene activity in a PRC sequence-dependent manner. These results suggested that PRC could exist bound to a repressor which was displaceable by other transcription factors such as HNF-1. Results obtained by transfection of HepG2 hepatoma cells with various PRB substitution mutant-luciferase gene fusion constructs indicated that the entire sequence of PRB was necessary for promoter activity. Consequently, the regulation of CYP1A3 expression is very complex, requiring a number of both positive and negative regulatory factors.

• BMB Reports
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• v.49 no.7
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• pp.394-399
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• 2016

Glioma-Associated Oncogene Homolog1 (Gli1) is known to be activated in malignant glioma; however, its downstream pathway has not been fully explained. The aim of this study was to explore the role of Gli1-Aquaporin1 (AQP1) signal pathway in glioma cell survival. Our data suggests that both Gli1 and AQP1 are upregulated in glioma tissues, as in comparison to in normal tissues. These up-regulation phenomena were also observed in glioma U251 and U87 cells. It was demonstrated that Gli1 positively regulated the AQP1 expression. By luciferase reporter gene and ChIP assay, we observed that this modulation process was realized by combination of Gli1 with AQP1 promotor. In addition, knock down of Gli1 by siRNA interference reduced the viability of glioma cells as well as suppressed cell metastasis. Also, the inhibitory effects of cell survival by silenced Gli1 were abrogated by AQP1 overexpression. In summary, glioma cell survival is a regulatory process and can be mediated by Gli1-AQP1 pathway.

• The Korean Journal of Nuclear Medicine
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• v.38 no.2
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• pp.111-114
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• 2004

Molecular imaging provides a visualization of normal as well as abnormal cellular processes at a molecular or genetic level rather than at a anatomical level. Conventional medical imaging methods utilize the imaging signals produced by nonspecific physico-chemical interaction. However, molecular imaging methods utilize the imaging signals derived from specific cellular or molecular events. Because molecular and genetic changes precede anatomical change in the course of disease development, molecular imaging can detect early events in disease progression. in the near future, through molecular imaging we can understand basic mechanisms of disease, and diagnose earlier and, subsequently, treat earlier intractable diseases such as cancer, neuro-degenerative diseases, and immunologic disorders. In beginning period, nuclear medicine started as a molecular imaging, and has had a leading role in the field of molecular imaging. But recently molecular imaging has been rapidly developed. Besides nuclear imaging, molecular imaging methods such as optical imaging, magnetic resonance imaging are emerging. Each imaging modalities have their advantages and weaknesses. The opportunities from molecular imaging look bright. We should try nuclear medicine continues to have a leading role in molecular imaging.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.4
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• pp.1575-1579
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• 2015

Some microRNAs (miRNAs) have been shown to act as oncogenes or tumor suppressor genes in human melanomas. miR-328 is upregulated in blood cells of melanoma patients compared to in healthy controls. This suggests a role for miR-328 in melanoma that warrants investigation. In this study, we demonstrated miR-328 levels to be dramatically decreased in human melanoma cell lines. Moreover, forced expression of miR-328 inhibited proliferation and induced G1-phase arrest of the SK-MEL-1 melanoma cell line. We identified TGFB2 as a direct target gene for miR-328 using a fluorescent reporter assay and western blotting. Levels of TGFB2 were dramatically increased in human melanoma cell lines and were inversely correlated with the miR-328 expression level. Our findings provide new insights into the mechanisms of human melanoma development, indicating that miR-328 has therapeutic potential for this disease.

• Biomolecules & Therapeutics
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• v.25 no.2
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• pp.165-170
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• 2017

Cordyceps bassiana is one of Cordyceps species with anti-oxidative, anti-cancer, anti-inflammatory, anti-diabetic, anti-obesity, anti-angiogenic, and anti-nociceptive activities. This mushroom has recently demonstrated to have an ability to reduce 2,4-dinitrofluorobenzene-induced atopic dermatitis symptoms in NC/Nga mice. In this study, we further examined phytochemical properties of this mushroom by column chromatography and HPLC analysis. By chromatographic separation and spectroscopic analysis, 8 compounds, such as 1,9-dimethylguanine (1), adenosine (2), uridine (3), nicotinamide (4), 3-methyluracil (5), 1,7-dimethylxanthine (6), nudifloric acid (7), and mannitol (8) were identified from 6 different fractions and 4 more subfractions. Through evaluation of their anti-inflammatory activities using reporter gene assay and mRNA analysis, compound 1 was found to block luciferase activity induced by $NF-{\kappa}B$ and AP-1, suppress the mRNA levels of cyclooxygenase (COX)-2 and tumor necrosis factor $(TNF)-{\alpha}$. Therefore, our data strongly suggests that compound 1 acts as one of major principles in Cordyceps bassiana with anti-inflammatory and anti-atopic dermatitis activities.

• Journal of Microbiology and Biotechnology
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• v.29 no.9
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• pp.1488-1493
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• 2019

The rising cases of multidrug-resistant Acinetobacter baumannii (Ab) and the lack of effective drugs call for quick attention. Here, based on a Tn7 transposon and Xer/dif system, we constructed a stable, selectable marker-free autoluminescent Ab capable of producing visible light without extra substrates. Utilization of this autoluminescent reporter strain has the potential to reduce the time, effort and costs required for the evaluation of activities of anti-Ab drug candidates in vitro.

• Radiation Oncology Journal
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• v.19 no.2
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• pp.171-180
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• 2001

Purpose : Expression of TIMP, intrinsic inhibitor of MMP, is regulated by signal transduction in response to genotoxins and is likely to be an important step in metastasis, angiogenesis and wound healing after ionizing radiation. Therefore, we studied radiation mediated TIMP expression and its mechanism in head and neck cancer cell lines. Materials and Methods : Human head and neck cancer cell lines established at Asan Medical Center were used and radiosensitivity $(D_0)$, radiation cytotoxicity and metastatic potential were measured by clonogenic assay, n assay and invasion assay, respectively. The conditioned medium was prepared at 24 hours and 48 hours after 2 Gy and 10 Gy irradiation and expression of TIMP protein was measured by Elisa assay with specific antibodies against human TIMP. hTIMP1 promoter region was cloned and TIMP1 luciferase reporter vector was constructed. The reporter vector was transfected to AMC-HN-1 and -HN-9 cells with or without expression vector Ras, then the cells were exposed to radiation or PMA, PKC activator. EMSA was peformed with oligonucleotide (-59/-53 element and SP1) of TIMP1 promoter. Results : $D_0$ of HN-1, -2, -3, -5 and -9 cell lines were 1.55 Gy, 1.8 Gy, 1.5 Gt, 1.55 Gy and 2.45 Gy respectively. n assay confirmed cell viability, over $94\%$ at 24hrs, 48hrs after 2 Gy irradiation and over 73% after 10 Gy irradiation. Elisa assay confirmed that cells secreted TIMP1, 2 proteins continuously. After 2 Gy irradiation, TIMP2 secretion was decreased at 24hrs in HN-1 and HN-9 cell lines but after 10 Gy irradiation, it was increased in all cell lines. At 48hrs after irradiation, it was increased in HN-1 but decreased in HN-9 cells. But the change in TIMP secretion by RT was mild. The transcription of TIMP1 gene in HN-1 was induced by PMA but in HN-9 cell lines, it was suppressed. Wild type Ras induced the TIMP-1 transcription by 20 fold and 4 fold in HN-1 and HN-9 respectively. The binding activity to -59/-53, AP1 motif was increased by RT, but not to SP1 motif in both cell lines. Conclusions : We observed the difference of expression and activity of TIMPs between radiosensitive and radioresistant cell line and the different signal transduction pathway between in these cell lines may contribute the different radiosensitivity. Further research to investigate the radiation response and its signal pathway of TIMPs is needed.