以肝癌細胞株HepG2探討缺氧狀態下PIAS2對於缺氧誘導因子-1α SUMO化機制

Abstract

肝癌在許多先進國家是癌症死亡的主要原因，而一直以來也高居台灣十大癌症死因首位，對國人健康影響甚鉅。肝癌的發展過程常常伴隨著缺氧狀態的產生，且肝癌是多血管型的腫瘤，缺氧狀態下其缺氧誘導因子-1α(HIF-1α)所主導的血管新生會促進肝癌腫瘤的發育。此外，臨床證據顯示肝癌病人組織SUMO-1的表現較正常組織為高，顯示SUMO化在肝癌癌化過程中扮演了一定的角色。缺氧誘導因子-1α，為一種對於氧分壓敏感的轉錄因子，在正常氧分壓下很快就會被降解，在缺氧狀態下的轉錄活性與穏定度受到多種轉譯後修飾(posttranslational modifications)所調控，其中包括SUMO化。SUMO化能改變細胞中分子的行為，諸如轉錄因子的活化或抑制、影響細胞中分子的穏定度、改變分子在細胞膜上的位置。而目前已知HIF-1α在缺氧狀態下會被SUMO化修飾，且SUMO化的循環中有E1活化酶(AOS1/UBA2)、E2銜接酶(UBC9)及SENP-1去SUMO酵素的參與，且SENP-1為HIF-1α下游基因，與HIF-1α之間存在著正迴饋的關係，但目前仍不清楚為何種SUMO E3黏合酶參與其中。近年來許多研究指出SUMO E3黏合酶－PIAS家族成員和腫瘤惡化與血管新生的相關性，其中PIAS2，又名PIASx(Protein Inhibitors of Activated STAT 2;PIAS2;PIASx)為PIAS SUMO E3連結酶家族成員之一，在正常肝臟組織中會高度表現，故本研究希望能夠透過進一步實驗，觀察當肝癌細胞處於缺氧微環境時，PIAS2是否參與在HIF-1α SUMO化過程中，以了解PIAS2在缺氧狀態下肝癌發育中所扮演的角色。本研究採用肝癌細胞株HepG2及HEK293T做為材料，以西方墨點法分析，利用缺氧系統培養箱或氯化亞鈷(II)所引起的缺氧狀態處理HepG2，發現其PIAS2的蛋白表現量並沒有改變；利用pcDNA3.1 -HA-HIF-1α對HEK293T進行暫時性轉染(transient transfection)，發現HIF1-α大量表現後並不會去影響PIAS2蛋白表現量；而利用免疫沈澱法觀察到細胞在缺氧狀態下不改變HepG2中PIAS2及SUMO-1蛋白表現量，且SENP-1被誘發的條件下，會促進PIAS2與HIF-1α之間的交互作用，也會增進HIF-1α與SUMO-1之間的交互作用；以PIAS2 shRNA慢病毒載體將HepG2的PIAS2進行靜默，觀察到缺氧狀態下PIAS2靜默HepG2和正常細胞相比，其HIF-1α與SUMO-1之間的交互作用程度會降低，且HIF-1α的穏定度也會下降，而在核質分離的結果中也發現，HIF-1α及SENP-1在核內的分布會減少。另外，在HRE promoter reporter assay方面，發現缺氧狀態下，在內生性PIAS2表現量靜默後，缺氧反應元件(hypoxia responsive element, HRE)活性會明顯地下降，此外利用RT-PCR也觀察到HepG2中HIF-1α下游基因VEGF及Glut-1，其mRNA表現量也有明顯減少的現象。總結以上結果，在缺氧狀態下HepG2中的PIAS2可藉由與HIF-1α交互作用並對其SUMO化，增加穏定度進而使HIF-1α進入核內。進核後SUMO型態的HIF-1α，會被SENP-1去SUMO化，而趨於穏定與HIF-1β結合至缺氧反應元件(hypoxia-resposive element, HRE)，使HRE活性上升，並轉錄活化下游基因VEGF及Glut-1之表現。本研究證明PIAS2於缺氧狀態訊息傳遞與下游反應的功能性重要角色。然而，其中可能牽涉其他SUMO E3黏合酶與轉譯後修飾作用參與則需要更進一步的實驗來探討相關機制。

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.