[논문]Identification of Differentially Expressed Genes (DEGs) by Malachite Green in HepG2 Cells

Kim, Youn-Jung; Song, Mee; Ryu, Jae-Chun

[국내논문] Identification of Differentially Expressed Genes (DEGs) by Malachite Green in HepG2 Cells 원문보기

Molecular & cellular toxicology, v.4 no.1, 2008년, pp.22 - 30

Kim, Youn-Jung (Cellular and Molecular Toxicology Laboratory, Korea Institute of Science and Technology) , Song, Mee (Cellular and Molecular Toxicology Laboratory, Korea Institute of Science and Technology) , Ryu, Jae-Chun (Cellular and Molecular Toxicology Laboratory, Korea Institute of Science and Technology)

Abstract ▼ AI-Helper

Malachite Green (MG), a toxic chemical used as a dye, topical antiseptic and antifungal agent for fish, is highly soluble in water, cytotoxic to various mammalian cells and also acts as a liver tumor promoter. In view of its industrial importance and possible exposure to human beings, MG possesses a potential environmental health hazard. So, we performed with HepG2, a human hepatocellular carcinoma cell line, to identify the differentially expressed genes (DEGs) related to toxicity of MG. And we compared gene expression between control and MG treatment to identify genes that are specifically or predominantly expressed by employing annealing control primer (ACP)-based $GeneFishing^{TM}$ method. The cytotoxicity $(IC_{20})$ of MG was determined above the $0.867{\mu}M$ in HepG2 cell for 48 h treatment. And the DEGs of MG were identified that 5 out of 6 DEGs were upregulated and 1 out of 6 DEGs was down-regulated by MG. Also, MG induced late apoptosis and necrosis in a dose dependent in flow cytometric analysis. Through further investigation, we will identify more meaningful and useful DEGs on MG, and then can get the information on mechanism and pathway associated with toxicity of MG.

Keyword

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가설 설정

This study examined whether MG has an influence on the expression of mRNA in HepG2, the human hepatocellular carcinoma cell line. The identification of differentially expressed genes (DEGs) induced by MG may assist in the identification of potential biomarker and may understand molecular toxicological mechanisms in HepG2 cells.

제안 방법

Unlike existing DEGs discovery method, ACPbase PCR method involves an ACP that has a unique tripartite structure in that its distinct 30- and 50-end portions are separated by a regulator facilitates the identification of DEGs from samples displaying low levels without generating false positives^9,16-18. In this study, GeneFishing^TM method applied to the ACPbased PCR method was employed in order to find DEGs responded by the effect of MG in HepG2 cells.
Polymerase chain reaction (PCR) amplification was conducted using the same GeneFishingTM DEG kits (Seegene) in 50 µL of reaction solution, consisting of 10X buffer without MgCl2, 25 mM MgCl2, 5 µM arbitrary ACPs, 10 µM dTACP2, 2 mM dNTP, 2.5 U Taq DNA polymerase (Genecraft, Ludinghausen, Germany), and 50 ng of first-strand cDNA, using a DNA Thermal Cycler 9600 (Perkin Elmer).
The thermal cycler was preheated to 94℃ before the tubes were placed in it. The program of PCR amplification was as follows; 1 cycle at 94℃ for 5 min, 50℃ for 3 min, and 72℃ for 1 min, 40 cycles at 94℃ for 40 s, 65℃ for 40 s, and 72℃ for 40 s, and 72℃ for 5 min. The DNA fragments produced by PCR were separated by electrophoresis on a 2% agarose gel.
The real time RT-PCR was performed by using a SYBR supermix kit (Bio-Rad), and running for 45 cycles at 95℃ for 20 s and 60℃ for 1 min. The PCR efficiency was examined by serially diluting the template cDNA and the melting curve data were collected to check the PCR specificity. Each cDNA sample was triplicated and the corresponding no-RT mRNA sample was included as a negative control.
In order to check whether MG induce apoptosis in HepG2 cells or not, the presence of apoptotic HepG2 cells using the flow cytometry assays was confirmed by double staining with propidium iodide and Annexin-V-FITC. Untreated controls maintained a relatively uniform distribution throughout the experiment.

이론/모형

867 µM (Figure 1). So, we used this concentration for GeneFishing^TM method.

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