鑑別蛋白質Shugoshin-1作為有潛力的肝癌治療之標靶

Identify protein Shugoshin-1 as a potential therapeutic target for hepatocellular carcinoma

王律涵(Wang, Lyu Han)

指導教授 ： 王慧菁

國立清華大學/生命科學院/分子與細胞生物研究所/博士(2015年)

摘要

Shugoshin-1(Sgo1)的功能在於保護著絲點上的cohesin以確保正確的染色體分離和維持基因組的完整性。有絲分裂期間，Plk-1磷酸化cohesin的亞單位SA2並導致cohesin離開染色體臂。相對的，著絲點的cohesin仍然停留於著絲點。原因是Sgo1與PP2A可以抵消Plk1對著絲點的cohesin的磷酸化。因此，著絲點的cohesin持續捆住姊妹染色體直到細胞進入anaphase。在本篇研究中，我們發現Plk1的活性不會影響Sgo1在有絲分裂期間的蛋白質表現和細胞內的定位。另一方面，我們在正常組織和轉型細胞株內偵測Sgo1異構體 (isoform) 的信使核糖核酸的表現。結果顯示轉型細胞株皆有表現Sgo1異構體的信使核糖核酸。除了胸線和睪丸，我們在其他正常組織中則無法偵測到Sgo1異構體的表現。而與永生細胞株RPE-1和NeHepLxHT比較，Sgo1蛋白會大量表現在轉型細胞株，如: HeLa、HuH-7、WRL-68、HepG2以及HepaRG。與周邊非肝癌樣本比較，我們也發現肝癌會大量表現Sgo1的信使核糖核酸和蛋白質。利用小分子干擾核糖核酸抑制Sgo1的表現導致了HeLa和HuH-7細胞的染色體缺失並使紡錘體裝配檢驗點 (spindle assembly checkpoint) 持續活性化。這個結果阻止細胞分裂的進行並引起細胞大量死亡於有絲分裂期。但永生細胞株RPE-1和NeHepLxHT並不受到抑制Sgo1的表現而大量死亡於有絲分裂期。另外，我們也發現在肝癌的周邊正常組織中，乙型肝炎病毒的表現與Sgo1蛋白的表現呈正相關。NeHepLxHT細胞株表現LHBs的細胞也有Sgo1大量表現的情況，並在有絲分裂的過程中伴隨染色體缺失。這些結果顯示Sgo1對於轉型細胞株和帶有LHBs蛋白的肝細胞在有絲分裂的過程中是重要的調控蛋白。這些結果顯示Sgo1可能是一個治療肝癌的新標靶，尤其是受到乙型肝炎病毒感染的肝癌。

關鍵字

有絲分裂 ； 肝癌 ； 乙型肝癌病毒 ； 標靶治療

並列摘要

Protein Shugoshin-1 (Sgo1) functions to protect centromeric cohesion and is essential for proper chromosome segregation and genomic integrity. During mitosis, Plk-1 phosphorylates SA2 subunit of cohesin, and thereby cohesin dissociates from chromosome arm. In contrast, centromeric cohesin is still kept at centromere due to the reason that Sgo1-protein phosphatase 2A (PP2A) counteracts Plk1-mediated phosphorylation at centromeric cohesin. Therefore, centromeric cohesin still tethers sister chromatids together until the onset of anaphase. In this study, we find that protein stability and localization of Sgo1 are independent of Plk1 activity during mitosis. In addition, we examined expression levels of Sgo1 isoforms in normal tissue cDNA and transformed cell lines by conventional reverse transcription-PCR. All Sgo1 isoforms are detected in the transformed cell lines, but not in most normal tissues, expect for thymus and testis. In contrast to hTERT-immortalized cell lines, we find that Sgo1 protein is highly expressed in transformed cell lines, such as HeLa, HuH-7, WRL-68, HepG2, and HepaRG. We also demonstrate that mRNA and the protein levels of Sgo1 significantly increased in HCC, compared with in adjacent non-HCC regions. The depletion of Sgo1 induces chromosome instability and mitotic cell death in HeLa and HuH-7 cells, as a result of spindle assembly checkpoints activation. This effect is not detected in hTERT-immortalized cell lines such as RPE-1 and NeHepLxHT. Notably, hepatitis B virus (HBV) is positively correlated with Sgo1 expression at non-HCC regions. Overexpression of viral large surface proteins (LHBS) increases Sgo1 expression on NeHepLxHT cells along with increased chromosome instabilities. These results demonstrated that Sgo1 is an important modulator for mitotic progression in transformed cells and hepatocytes carrying HBV-LHBS protein. We suggest that Sgo1 is a promising and novel therapeutic target for HCC, especially those related to HBV infection.

並列關鍵字

Mitosis ； Shugoshin-1 ； Hepatocellular carcinoma ； Targeted therapy ； Hepatitis b virus

參考文獻

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