NFE2L1의 미토콘드리아 손상 반응성과 간암세포주의 침윤활성 조절 연구
Mitochondrial dysfunction increases Hepatoma cell invasiveness via NFE2L1 expression
- 주제(키워드) mitochondria , invasiveness
- 발행기관 아주대학교
- 지도교수 윤계순
- 발행년도 2017
- 학위수여년월 2017. 2
- 학위명 석사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000024212
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Mitochondrial dysfunction is an important metabolic feature in human cancer. However, underlying mechanisms how mitochondrial dysfunction affects tumorigenesis remain unclear. The current study focused on the role of NFE2L1 transcription factor which was previously identified as a mitochondrial defect-responsive gene in hepatoma cell invasiveness. NFE2L1 expression is increased in SNU354 and SNU423 hepatoma cells which have mitochondrial defects and high invasion activity. Knockdown of NFE2L1 decreased hepatoma cell invasion activity in SNU354 and SNU423 cells, implying that NFE2L1 is a key regulator of invasion activity. Next, we investigated how NFE2L1 expression is regulated by mitochondrial dysfunction. SNU354 and SNU423 cells showed high cytosolic Ca2+ and reactive oxygen species (ROS) levels and both increased cytosolic Ca2+ and ROS levels were involved in NFE2L1 expression. Analysis of transcription factor binding sites within -2800 to +200 NFE2L1 promoter region using TRANSFAC program identified 11 potential transcription factors to regulate NFE2L1 transcription. Among them, sterol regulatory element-binding protein 1 (SREBP1) was found to regulate NFE2L1 expression in SNU354 cell. In conclusion, our results suggest that NFE2L1 is one of key transcription regulators to control hepatoma cell invasiveness and its transcription is regulated by mitochondria driven Ca2+ or ROS signaling. Also, SREBP1 may be an upstream transcription factor of the NFE2L1 transcription. These findings emphasize the involvement of NFE2L1 in hepatocellular carcinoma (HCC), providing its potential role as a therapeutic target of HCC, and expand our knowledge of mitochondrial defect-mediated mitochondrial retrograde signaling in tumorigenesis.
more목차
I. INTRODUCTION 1
II. MATERIALS AND METHODS 6
1. Cell culture and cell growth rate 6
2. Western blot analysis 6
3. Real-time reverse transcription-polymerase chain reaction (qPCR) 7
4. Transfection of siRNAs 8
5. Cell invasion assay 8
6. Construction of the promoter-luciferase reporter plasmid and promoter assay 9
7. Gene expression profiling and data analysis 9
8. Estimation of intracellular ROS levels 10
9. Endogenous cellular oxygen consumption rate 10
10. Infection of recombinant adenovirus 11
11. Generation of cell clones stably expressing NFE2L1 short hairpin RNAs 11
12. Deglycosylation of protein 11
III. RESULTS 12
1. Mitochondrial respiratory defect is associated with invasion activity in hepatoma cells. 13
2. Increased expression of NFE2L1 is associated with mitochondrial defect in hepatoma cell line. 13
3. Mitochondrial dysfunction induces NFE2L1 expression, not NFE2L2 or NFE2L3 in hepatoma cells. 15
4. NFE2L1 expression regulates invasion activity in hepatocellular carcinoma cells harboring defective mitochondria 19
5. NFE2L1 is regulated by cytosolic calcium levels and ROS in hepatocellular carcinoma. 22
6. Analysis of potential transcription factor binding sites in NFE2L1 promoter region. 25
7. NFE2L1 was regulated by SREBP1 in liver cells 30
8. Mitochondrial defect increased NFE2L1 promoter activity in liver cells. 36
IV. DISCUSSION 39
V. REFERENCES 41
국문요약 45
