간암세포주에서 나타나는 LDHB 발현저하에 의한 미토콘드리아의 호흡 활성 조절기전 연구
Regulation of mitochondrial respiration by LDHB suppression in hepatoma cell lines
- 주제(키워드) Mitochondrial dysfunction , Aerobic glycolysis , LDHB (lactate dehydrogenase B) , PDH (pyruvate dehydrogenase) , Lactic acidosis , Hepatoma cell
- 발행기관 아주대학교
- 지도교수 윤계순
- 발행년도 2015
- 학위수여년월 2015. 8
- 학위명 석사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000020561
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Increase in aerobic glycolysis and mitochondrial dysfunction are important metabolic features in human cancers. However, it still remains unclear how these phenomena are developed during tumorigenesis. To maintain aerobic glycolysis, continuous generation of NAD+ by lactate dehydrogenase (LDH) with conversion of pyruvate to lactate is essential. Functional LDH is homo- or heterotetramers assembled from two types of subunit with LDHA and LDHB gene product. Recently, we reported that LDH5 isoenzyme formed by LDHB suppression is involved in increased glycolytic lactate production and mitochondrial respiratory defects in hepatoma cells. In this study, we aimed to investigate how LDHB suppression is linked with mitochondrial respiratory dysfunction. When LDHB expression was suppressed by siRNA-mediated knockdown, cellular oxygen consumption rate was significantly decreased without clear decrease in expressions of respiratory proteins. These results imply that LDHB suppression may control mitochondrial respiration through post translational modification or respiratory subunits or control of metabolites. Thus, we focused on pyruvate dehydrogenase (PDH) which is a key regulatory enzyme linking glycolysis and mitochondrial activity by controlling TCA cycle. Interestingly, LDHB knockdown effectively increased phosphorylation of PDH, indicating its inactivation. Treatment of lactate without LDHB knockdown increased PDH phosphorylation with lowering pH. This result implies that PDH phosphorylation is correlated with acidification, but is not lactate-specific event. Collectively, our results suggest that lactate-mediated PDH inactivation is the key mechanism to induce mitochondrial dysfunction in LDHB-suppressed hepatoma cell.
more목차
I. INTRODUCTION 1
II. MATERIAL AND METHODS 5
A. Cell cultures and cell growth rate 5
B. Western blot analysis 5
C. Transfection of siRNAs 6
D. LDH isoenzyme in gel activity 7
E. Lactate level in cultured medium 7
F. Real-time reverse transcription-polymerase chain reaction (RT-PCR) 8
G. Mitochondrial membrane potential 8
H. Endogenous cellular oxygen consumption rate 9
I. Chemicals 9
III. RESULTS 10
1. Decreased LDHB expression-mediated lactic acidosis is associated with mitochondrial
respiratory activity. 10
2. LDHB suppression is an upstream event of decreased mitochondrial respiration. 13
3. LDHB knockdown induces PDK-mediated PDH phosphorylation without HIF1-PDK1 induction. 17
4. Lactic acidosis increases PDH phosphorylation. 22
5. LDHB-suppression-induced PDH phosphorylation is mediated through ERK activation. 27
6. Lactic acidosis delays cell growth with morphological changes. 30
IV. DISCUSSION 34
REFERENCES 36
국문요약 41
