미토콘드리아 기능이상이 간암세포에 미치는 영향: mtOGG1과 NUPR1의 역할 규명
Effect of mitochondrial respiratory defect on hepatocellular carcinoma: Involvement of mtOGG1 and NUPR1
- 발행기관 아주대학교
- 지도교수 윤계순
- 발행년도 2014
- 학위수여년월 2014. 8
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000017778
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Many solid tumor cells exhibit mitochondrial respiratory impairment. However, the mechanisms of such impairments in cancer development remain unclear. In this study, to investigate an effect of mitochondrial dysfunction on hepatocellular carcinoma, the two projects were studied. The first project was to prove the involvement of mitochondrial DNA repair enzyme in hepatoma cell growth. To address this, it was employed several SNU hepatoma cells, which had been classified as harboring mitochondrial respiratory defects or not in a previous study. Interestingly, SNU cells with declined mitochondrial respiratory activity showed decreased expression of mitochondrial 8-oxoguanine DNA glycosylase/lyase (mtOGG1), a mitochondrial DNA repair enzyme; similar results were obtained with human hepatocellular carcinoma tissues. Among several OGG1-2 variants with a mitochondrial-targeting sequence (OGG1-2a, -2b, -2c, -2d, and -2e), OGG1-2a was the major mitochondrial isoform in all examined hepatoma cells. Interestingly, hepatoma cells with low mtOGG1 levels showed delayed cell growth and increased intracellular reactive oxygen species (ROS) levels. Knockdown of OGG1-2 isoforms in Chang cells, which have active mitochondrial respiration with high mtOGG1 levels, significantly decreased cellular respiration and cell growth, and increased intracellular ROS. Overexpression of OGG1-2a in SNU423 cells, which have low mtOGG1 levels, effectively recovered cellular respiration and cell growth activities, and decreased intracellular ROS. Taken together, these results suggest that mtOGG1 plays an important role in maintaining mitochondrial respiration, thereby contributing to cell growth of hepatoma cells. The second project was to identify major genes to control hepatoma cell invasiveness in response to mitochondrial respiratory defects. To address this, cDNA microarray was performed using SNU hepatoma cells with and without mitochondrial defects. Interestingly, functional enrichment analysis showed that 1423 commonly up-regulated genes in the hepatoma cells with mitochondrial defects were mainly linked with cell migration and actin organization. To further identify genes that are specifically up-regulated in response to mitochondrial respiratory defects, additional mitochondrial defects models were employed using respiratory inhibitors and mitochondrial DNA depletion (Rho 0). Among 10 genes commonly up-regulated in the three models, three transcription factors, NUPR1, NFIX, and NFE2L1, were further evaluated. All three transcription factors were involved in hepatoma cell invasiveness. Interestingly, NFIX induction was mediated by ROS, whereas NUPR1 and NFE2L1 expressions were mediated by Ca++ increase. Next, granulin (GRN) was identified as a potential downstream target of NUPR1 by employing the cDNA microarray analysis of siRNA-mediated NUPR1-depleted SNU354 cell. Finally, GRN was proved to be a critical molecule to regulate hepatoma cell invasiveness. Taken together, these results suggested that NUPR1 is a key mitochondrial retrograde Ca++ signal-responsive transcription factor to control hepatoma cell invasiveness.
more목차
I. INTRODUCTION 1
II. Decreased mitochondrial OGG1 expression is linked to mitochondrial defects and delayed hepatoma cell growth 11
A. INTRODUCTION 12
B. MATERIALS AND METHODS 14
1. Cell cultures, cell growth rate and tumor samples 14
2. Reverse transcription-polymerase chain reaction (RT-PCR) 14
3. Construction of mtOGG1 cDNA plasmids and transfection of cDNA plasmids and siRNAs. 15
4. Endogenous cellular oxygen consumption rate 15
5. Estimation of intracellular and mitochondrial ROS level 16
6. Subcellular fractionation 16
7. Total genomic DNA isolation and sequencing of mitochondrial DNA fragments 17
8. Southern blot analysis of mtDNA 17
9. Western blot analysis 18
C. RESULTS 19
1. Decreased mtOGG1 expression is associated with mitochondrial dysfunction in hepatoma cells and tissues. 19
2. OGG1-2a is the major mitochondrial-targeting OGG1 in Chang-L and hepatoma cells. 25
3. mtOGG1 suppression decreases mitochondrial respiration and cell growth rate, and increases intracellular ROS level. 30
4. mtOGG1 overexpression recovered mitochondrial respiration and cell growth rate, and decreased intracellular ROS level in SNU423 cells. 34
D. DISCUSSION 37
III. Key mitochondrial retrograde signal-responsive transcription factors to control hepatoma cell invasiveness 39
A. INTRODUCTION 40
B. MATERIALS AND METHODS 42
1. Cell cultures, cell growth rate and tumor samples 42
2. Production of recombinant lentiviruses harboring target shRNAs and generation of cell clones stably expressing target shRNAs 42
3. Construction of recombinant cDNA plasmids and transfection of cDNA plasmids and siRNAs 43
4. Measurement of cellular oxygen consumption rate 44
5. Measurement of intracellular ROS and calcium levels 44
6. Cell invasion assay 44
7. Gene expression profiling and data analysis 45
8. Real-time reverse transcription-polymerase chain reaction (RT-PCR) 46
C. RESULTS 47
1. Ten genes were identified as possible mitochondrial retrograde signal-responsive genes to be involved in hepatoma cell invasion activity. 47
2. Commonly up-regulated three transcription factors are truly involved in hepatoma cell invasion activity. 54
3. NFIX expression is controlled by ROS. 57
4. NUPR1 and NFE2L1 are regulated by cytosolic Ca++ increase. 61
5. Granulin is a key downstream target molecule induced by NUPR1, thereby controlling hepatoma cell invasion activity. 66
D. DISCUSSION 71
IV. CONCLUSION 74
V. REFFERENCE 75
국문요약 84
