| Summary: | 碩士 === 國立臺灣大學 === 毒理學研究所 === 99 === Hepatocellular carcinoma(HCC), which posed dangerous threat to human health, was the leading cause of death among cancers worldwide, including Taiwan. The development of HCC was always along with hypoxic conditions. Moreover, HCC was a hypervascular tumor and hypoxia-inducible factor-1α(HIF-1α)-mediated angiogenesis under hypoxia played an important role in its progression. Additionally, clinical studies have shown that the expression level of SUMO-1 was more higher in HCC tissue than non-neoplastic tissue, which suggested that SUMOylation plays a key role in the development of HCC. HIF-1α was a transcription factor that was rapidly degraded during normoxia and its ability to transactivate downstream genes and stability of HIF-1α was tightly regulated by many posttranslational modifications under hypoxia, including SUMOylation. SUMOylation could regulate diverse cellular functions, including transcription, nuclear translocation, and stress response. Previous studies have shown HIF-1α was modified by SUMO-1 under hypoxic conditions. In the process of HIF-1α SUMOylation, E1-AOS1/UBA2, E2-UBC9 and SENP-1 were involved. SENP-1, which was also a target gene of HIF-1α, could regulate HIF-1α by positive feedback under hypoxia. Nevertheless, how HIF-1α became SUMOylated by which SUMO E3 ligases during hypoxia is still unknown. According to recent research, there was emerging evidence showing that several members of PIAS(Protein Inhibitors of Activated STAT) SUMO E3 ligase family were possibly correlated to tumorogenesis and angiogenesis. PIAS2, a member of PIAS protein family, was highly expressed in liver. The aim of this study was to investigate the correlation of SUMOylation between HIF-1α and PIAS2 in human hepatocarcinoma cell line(HepG2). Treatment of 0.5% oxygen in hypoxia chamber and CoCl2 could not alter the protein level of PIAS2 in HepG2 cells. Molecular approaches to overexpress HIF-1α protein expression in HEK293T cells did not affect the protein level of PIAS2. Additionally, hypoxic conditions could enhance not only the interaction between PIAS2 and HIF-1α but also SUMO-1 and HIF-1α by Immunopreciptation(IP) in HepG2 cells on condition that SENP-1 could be induced. Besides, we used PIAS2 shRNA to silence the endogenous PIAS2 expression in HepG2 cells, and we subsequently found that the interaction between SUMO-1 and HIF-1α, the nuclear translocation of HIF-1α and SENP-1 and HRE activity were significantly reduced in PIAS2 silenced HepG2 cells compared to wild type cells under hypoxia. Furthermore, mRNA level of hypoxia-responsive genes such as VEGF and Glut-1 were significantly decreased in PIAS2 silenced HepG2 cells under hypoxia. Overall, we concluded that PIAS2 could play a key role in the process of HIF-1α nuclear translocation and transactivation activity via SUMOylating HIF-1α under hypoxic conditions. This study demonstrated that PIAS2 may play a key role in the hypoxia-mediated signaling and downstream gene expression. However, we will further confirm if there is any other SUMO E3 ligase and posttranslational modification involved in the mechanism of HIF-1α SUMOylation under hypoxia.
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