Journal of the Korea Convergence Society (한국융합학회논문지)

Korea Convergence Society (한국융합학회)
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The Convergence Analysis of Microarray-Based Gene Expression by Difference of Culture Environment in Human Oral Epithelial Cells

구강상피세포의 배양환경의 차이에 의한 마이크로어레이 기반 유전자 발현의 융복합 분석

  • Son, Hwa-Kyung (Department of Dental Hygiene, Yeungnam University College)
  • 손화경 (영남이공대학교 치위생과)
  • Received : 2019.03.12
  • Accepted : 2019.04.20
  • Published : 2019.04.28
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Abstract

This study was analyzed about the relationship between culture microenvironment and cell differentiation of HPV 16 E6/E7-transfected immortalized oral keratinocyte(IHOK). By the alteration of culture environment, IHOK-EF and IHOK-EFKGM were obtained, and the modulation of cell properties was observed by cell proliferation assay, immunofluorescence, microarray, and quantitative real-time PCR analysis. IHOK-EF losed the properties of epithelial cells and obtained the properties of mesenchymal cells, and in the result of microarray analysis, genes related to the inhibition of differentiation such as IL6, TWIST1, and ID2 were highly expressed in IHOK-EF. When the culture environment was recovered to initial environment, these changes were recovered partially, presenting the return of genes involved in the inhibition of differentiation such as IL6, and ID2, particularly. This study will contribute to understand adjustment aspect for cell surviving according to the change of culture microenvironment in the study for determining the cell characteristic, and facilitate therapeutic approach for human disease by applying surviving study according to the change of cancer microenvironment.

이 연구는 HPV 16 E6/E7 도입 불멸화 구강상피세포의 배양 미세환경과 세포 분화간의 관계를 분석하였다. 배양환경을 변화시켜서 IHOK-EF 세포와 IHOK-EFKGM 세포를 얻었고, 이들 세포의 특성변화를 세포증식분석, 면역형광분석 및 마이크로어레이와 실시간 정량 PCR분석으로 알아보았다. IHOK-EF 세포는 상피세포의 특성을 상실하고 간엽세포의 특성을 획득하였고, 마이크로어레이 분석결과, 분화억제 유전자인 ID2, IL6, TWIST1이 과발현 되었다. 이러한 변화는 초기의 배양환경으로 회복되었을 때, 특별히, ID2와 IL6에서 유전자발현의 복귀를 나타내면서 세포의 특성이 부분적으로 회복되었다. 이 연구는 세포의 특성을 결정하는 연구에서 배양 미세환경의 변화에 따른 세포의 생존을 위한 적응양상을 이해하는데 공헌할 것이며, 향후, 암세포의 미세환경변화에 따른 생존연구에 적용하여 질병에 대한 치료적 접근을 가능하게 할 것이다.

Keywords

OHHGBW_2019_v10n4_81_f0001.png 이미지

Fig. 1. The alteration of IHOK by change of culture conditions (A) Control IHOK-KGM cells were cultured in two media. a, Control IHOK-KGM cells, b, IHOK-KGM cells were cultured in EF media for 90days, c, IHOK-EF cultured for 90days were re-cultured in KGM for 120 days. (B) The cell growth rate of control IHOK-KGM and two altered cell lines was analyzed.

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Fig. 2. The Analysis of Restoration from IHOK-EF to IHOK-KGM (A) RT-PCR was analized by EMT(E-cadherin, vimentin, snail), differentiation(involucrin,) and cell cycle regulation(cyclin D1) markers. (B) Cells were stained by vimentin and cytokeratin antibodies during 2 hr, respectively, for analyss of Immunofluoresence

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Fig. 3. Hierarchical clustering for more than doubled changed genes in Microarray data Three kinds of IHOKs cultured by two media were analyzed by microarray. Expressed genes were identified by comparisons of KGM cells to EF cells as well as KGM cells to EFKGM cells. In the data, red groups show upregulated genes and green groups show downregulated genes in comparison with KGM contol groups. Data show at least more than doubled difference among P value<0.05.

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Fig. 5. Validation of micoarray data for differentiation inhibitors by real time PCR mRNA expression of ID2, IL6, and TWIST1 was verified by real time PCR in the three cell lines.

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Fig. 4. Hierarchical clustering of more than doubled changed genes for cell differentiation Up-regulated or down-regulated genes, which expressed the highest intensity among returned genes more than doubled from genes changed more than doubled between IHOK-KGM control cells and IHOK-EF cells for cell differentiation, were shown.

Table 1. List of genes which inhibit epithelial differentiation in comparison for IHOK-KGM cells to IHOK-EF-cells and IHOK-KGM cells to IHOK-EFKGM cells among changed genes more than 3 fold.

OHHGBW_2019_v10n4_81_t0001.png 이미지

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