자궁경부암세포에서 방사선조사시 차등 발현되는 유전자 동정
Identification of Differentially Expressed Radiation-induced Genes in Cervix Carcinoma Cells Using Suppression Subtractive Hybridization
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김준상/Kim JS
이영숙/이증훈/이웅희/서은영/조문준/Lee YS/Lee JH/Lee WH/Seo EY/Cho MJ
KMID : 0859320050230010043
Abstract
목적: 자경경부암세포에서 polymeric chain reaction (PCR)원리를 이용한 suppression subtractive hybridization (SSH) 방법으로 방사선조사 시 차등 발현되는 유전자를 동정하고자 하였다.
대상 및 방법: 자궁경부암세포주인 HeLa 세포주에 방사선조사 전과 후 총 RNA와 poly (A)+ mRNA를 분리하였다. SSH방법으로 forward 및 reverse-subtracted cDNA libraries를 만들었다. 차등 발현된 유전자를 screening하기 위해 reverse Northern blotting (dot blot analysis)을 이용하여 각각의 library에서 88개의 클론을 선택하였고 Nothern blotting으로 확인 후 sequencing하였다.
결과: screening상 176개 클론 중 forward-subtracted library에서 10개의 유전자가 reverse-subtracted library에서 9개의 유전자가 동정되었다. forward-subtracted library로부터 3개의 유전자가 Northern blotting에 의하여 확인되었고 이중 telomerase catalytic subunit and sodium channel-like protein 유전자와 1개의 ESTs (expressed sequence tags) 유전자가 방사선선량에 따라 증가하였다.
결론: 본 연구를 통해 자궁경부암세포주에서 방사선에 의해 유도되는 유전자를 SSH 방법을 통해 동정할 수 있었다. 그러나 이러한 유전자가 어떤 생물학적인 기능을 갖고 있는지에 대한 계속적인 연구가 필요하다.
Purpose: A number of genes and their products are induced early or late following exposure of cells to ionizing radiation. These radiation-induced genes have various effects on irradiated cells and tissues. Suppression subtractive hybridization (SSH) based on PCR was used to identify the differentially expressed genes by radiation in cervix carcinoma cells.
Materials and Methods: Total RNA and poly (A)+ mRNA were isolated from irradiated and non-irradiated HeLa cells. Forward- and reverse-subtracted cDNA libraries were constructed using SSH. Eighty-eight clones of each were used to randomly select differentially expressed genes using reverse Northern blotting (dot blot analysis). Northern blotting was used to verify the screened genes.
Results: Of the 176 clones, 10 genes in the forward-subtracted library and 9 genes in the reverse-subtracted library were identified as differentially expressed radiation-induced genes by PCR-select differential screening. Three clones from the forward-subtracted library were confirmed by Northern blotting, and showed increased expression in a dose-dependent manner, including a telomerase catalytic subunit and sodium channel-like protein gene, and an ESTs (expressed sequence tags) gene.
Conclusion: We identified differentially expressed radiation-induced genes with low-abundance genes with SSH, but further characterization of theses genes are necessary to clarify the biological functions of them.
키워드
Cervix carcinoma; Suppression subtractive hybridization; HeLa cells; Radiation-induced genes;자궁경부암;HeLa 세포; 방사선유도유전자
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