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구강암세포 KB에서 Neuron Restrictive Silencer Factor siRNA에 의한 유전자 발현

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Author(s)
전우진
Issued Date
2011
Abstract
아연집게 단백질로 알려진 neuron restrictive silencer factor (NRSF)는 또한 RE-1 silencing transcription factor (REST)로 불리 우며, 비신경세포에서 neuron restrictive silencer element (NRSE)를 포함한 신경관련 유전자의 발현을 억제한다. 이는 비신경세포 뿐만 아니라 신경 전구세포에서도 발현 하지만 유사분열 후 신경에서는 발현되지 않는다. 그러므로 NRSF의 발현은 신경세포의 발달에 관련된 역할을 하며 신경관련 유전자의 발현을 유도함으로써 신경세포의 형성에 중요한 역할을 한다. 최근 들어 NRSF는 성숙된 신경세포에서 뿐만 아니라 암세포에서도 그 발현이 확인되었다. 그러나 구강암세포에서는 NRSE/NRSF의 조절 기전이 알려져 있지 않고 있다. 본 연구실에서는 선행연구로 NRSF가 구강암세포의 성장에 조절에 중요한 역할을 하고 있음을 확인하였고 NRSF의 발현을 siRNA를 이용하여 억제함으로써 구강암세포의 성장을 저해하고 있음을 확인하였다.
본 연구에서는 구강암세포 KB세포에서 NRSE/NRSF에 의한 조절 체계를 이해하기 위해 siRNA를 이용하여 KB세포의 NRSF의 발현을 억제한 후, microarray 분석을 통하여 유전자의 발현을 확인하였다. 다양한 유전자의 분석을 확인한 경과, 117개의 유전자가 그 발현이 증가하였고, 215개의 유전자가 발현이 억제되었다. 대부분의 발현이 증가된 유전자들은 signal transduction, cell communication, cell cycle, 그리고 apoptosis에 관여하는 유전자였으며 발현이 감소된 유전자들은 주로 RNA processing, neurogenesis, transcription factor activity, 그리고 synaptogenesis에 관여하고 있음을 확인하였다. 흥미로운 사실은 NRSF가 전사조절 억제자로 알려져 있으나 구강암세포에서는 NRSF의 siRNA에 의해 200개 이상의 유전자가 그 발현이 감소하고 있었다. 또한 50% 이상의 유전자가 번역이나 ribosomal 단백질의 복합체를 이루는 유전자로 확인 할 수 있었다.
본 연구결과로 구강암세포에서 NRSF를 통한 새로운 성장 조절 기전을 확인할 수 있는 유전자들을 선별할 수 있었고 더 나아가, 이들 유전자의 조절기전을 밝혀 새로운 기전을 확립 할 수 있을 것이다.|A zinc finger protein known as neuron restrictive silencer factor (NRSF) or the RE-1 silencing transcription factor (REST) binds to the neuron restrictive silencer element (NRSE) and silences expression of genes containing NRSEs in nonneural cells. NRSF is expressed ubiquitously in nonneural cells and in neuronal precursors but not in post-mitotic neurons. Threfore, down-expression of NRSF is likely to be a key event in neural development in that it releases silencing and allows expression of several genes that important for the establishment of the phenotype of neural cells. It has been reported that NRSF is highly expressed in many aggressive types of cancer cells as well as in mature neurons. However, the function of NRSE/NRSF system has been unknown in KB human oral cancer cells, completely. Previous study in our lab, NRSF is important factor to regulate the cell proliferation of KB human oral cancer cells. Cell growth was significantly inhibited by repression of NRSF expression using the NRSF specific target siRNA treatment in KB cells.
In this syudy, to understand the NRSE/NRSF regulatory network in KB cells, we performed the microarray analysis by NRSF specific targeted siRNA treatment in KB human oral cancer cells. The expressiom profiles of several genes were further validated in the NRSF siRNA treated KB cells. 117 genes were found to be upregulated and 215 genes downregulated in the NRSF siRNA treated KB cells by microarray analysis. The most of upregulated genes were involved in signal transduction, cell communication, cell cycle, and apoptosis, and downregulated genes were involved in RNA processing, neurogenesis, transcription factor activity, and synaptogenesis. Interestingly, although NRSF has been known as a transcriptional repressor to silence neuronal gene, out data shown that more than 200 genes were downregulated by NRSF siRNA treated KB cells. Specially, 50 % of these genes are involved in translational regulation and consist of ribosomal complex proteins.
As the results, the genes identified in this screen represent novel control pathway via NRSF expression in KB oral cancer cells. Further investigation will be need to define the mechanism of theirs gene regulation by NRSF expression in KB human oral cancer cells.
Alternative Title
Gene expression by neuron restrictive silencer factor siRNA in KB human oral cancer cells
Alternative Author(s)
Woo-Jin Jun
Affiliation
치의학과 구강악안면외과학
Department
일반대학원 치의학과
Advisor
김춘성
Awarded Date
2012-02
Table Of Contents
초 록 ⅲ

Ⅰ. INTRODUCTION

Ⅱ. MATERIALS & METHODS 4
Ⅱ-1. Cell culture 4
Ⅱ-2. siRNA and transfection 4
Ⅱ-3. RNA preparation 4
Ⅱ-4. DNA microarray analysis 5
Ⅱ-5. Microarray data analysis 5

Ⅲ. RESULTS 7
Ⅲ-1. Gene expression profile in NRSF-siRNA treated KB cells 7
Ⅲ-2. Gene expression data of microarray analysis by NRSF siRNA treated KB human oral cancer cells

Ⅳ. DISCUSSION 19

Ⅴ. REFFERENCES 23

ABSTRACT 32


LIST OF TABLES


Table 1. Upregulated gene categories by NRSF siRNA treated KB cells 8

Table 2. Downregulated gene categories by NRSF siRNA treated KB cells 9

Table 3. Upregulated genes of microarray analysis by NRSF siRNA 12

Table 4. Downregulated genes of microarray analysis by NRSF siRNA 15


LIST OF FIGURES


Fig. 1. Visul dis play of the cluster analysis for NRSF siRNA transfected and control cells 29

Fig. 2. Two-dimensinal scatterplot analysis of gene expression values for all genes on the NRSF siRNA transfected KB cells and control cells from the microarray 30

Fig. 3. Structure of human ribosomal complex and ribosomal related genes 31
Degree
Doctor
Publisher
치의학과
Citation
전우진. (2011). 구강암세포 KB에서 Neuron Restrictive Silencer Factor siRNA에 의한 유전자 발현.
Type
Dissertation
URI
https://oak.chosun.ac.kr/handle/2020.oak/9303
http://chosun.dcollection.net/common/orgView/200000256645
Appears in Collections:
General Graduate School > 4. Theses(Ph.D)
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  • Embargo2012-02-02
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