SIRT1 displays anticancer potential against advanced hepatocellular carcinoma with loss of p53 function

다국어 초록 (Multilingual Abstract)

Background: Hepatocellular carcinoma (HCC) is a highly malignant tumor with a poor prognosis. SIRT1 (silent mating-type information regulation 2 homologue 1) is a class III histone deacetylase that is implicated in gene regulations and stress resistance. Detection of SIRT1 is considered a useful predictor of prognosis in some carcinomas. However, its interpretation and clinical significance remains to be determined in hepatocellular carcinoma. Methods: A retrospective study was performed on the clinicopathological features of HCC from surgical cases. Protein expression level was detected by western-blotting. p53 mutation was identified by sequencing. Results: In this study, we found that SIRT1 was a strong and independent predictor for better prognosis in patients of advanced HCC with loss of p53 function. Down regulation of SIRT1 by shRNA consistently stimulated and reduced the cell growth and proliferation of PLC5 cells (mutation on 249 site of p53) and HepG2 cells (wild-type p53) respectively in vitro or in vivo. SIRT1 silencing caused suppression of AMP-activated protein kinase (AMPK) activity and subsequently enhanced mammalian target of rapamycin (mTOR) activity, resulting in induction of activity of S6 Kinase 1 (S6K1) in PLC5 but not in HepG2 cells in vitro and in vivo. Consistently, we detected a significant positive correlation between SIRT1 and phosphor-AMPK expression levels in a cohort of advanced HCC specimens with loss of p53 function, and the combination of these two parameters was a powerful predictor of prognosis for advanced HCC patients with loss of p53 function. Conclusions: SIRT1 displays an anti-carcinogenic role through AMPK-mTOR pathway in HCC with loss of p53 function. However, in HCC with normal p53, SIRT1 plays its role as a tumor promoter via its classical p53 pathway. The decision of the role following SIRT1 activation is greatly influenced by loss or normality of p53 function.

목차 (Table of Contents)

• 1. INTRODUCTION 1
• 2. MATERIALS AND METHODS 2
• 2.1 Patients
• 2.2 Western blotting analysis
• 2.3 Identification of p53 function by sequencing in HCC specimens
• 2.4 Cell culture
• 2.5 Lentivirus expressing shSIRT1 transfection
• 2.6 Retrovirus expressing wide type and mutant type p53
• 2.7 Cell growth and proliferation assay
• 2.8 Colony formation assay
• 2.9 Tumor xenograft assay
• 2.10 Immunohistochemistry (IHC)
• 2.11 Statistical analysis
• 3. RESULTS 8
• 3.1 Identification of p53 function by sequencing in HCC specimens
• 3.2 SIRT1 expression in HCC specimens
• 3.3 HCC stage specific and p53 function associated evaluation of SIRT1 in predicting patients’ survival
• 3.4 The role of SIRT1 in p53 pathway
• 3.5 The role of SIRT1 in AMPK-mTOR pathway
• 3.6 The role of SIRT1 in Hep3B cells expressing wide or mutant type p53
• 3.7 Correlation between SIRT1 and phosphor-AMPK expression in advanced HCC specimens
• 3.8 Effect of SIRT1 silencing on cell proliferation and cell colony formation in vitro
• 3.9 Effect of SIRT1 knockdown on tumor growth in vivo
• 4. DISCUSSION 43
• 5. REFERENCES 49
• 6. 국문초록 54