Pim1-RBMY交互作用之拮抗胜肽對於肝癌細胞活性之研究

Abstract

肝細胞癌為全世界發生普及且致死率較高的癌症之一，其中男性的好發率又較女性高，由於目前肝癌第一線藥物Sorafenib副作用較大，為達到更好的療效仍需找尋更好的治療方法。在先前研究中發現，Y染色體上RNA結合模體蛋白 (RBMY) 原先僅表現在精子細胞及發育中的肝細胞中，但卻在男性肝細胞癌患者的肝癌組織中卻異常活化及表現。RBMY蛋白帶有核醣核酸識別模體 (RRM) 及四段由絲胺酸、精胺酸、甘胺酸、酪胺酸的重複片段 (SRGY boxes)，然而RBMY蛋白僅N端RRM區域具有結構，SRGY區域為固有無序化胺基酸 (intrinsically disordered amino acids) 序列，序列靈活度高並且易與多種蛋白結合，藉此調控生物功能，但其序列並沒有固定三級結構，不易作為藥物設計之標靶，因此我們針對RBMY的上游激酶Pim1進行研究，藉由找出可與Pim1結合之特定序列，並以RBMY的胺基酸序列設計多段短片段胜肽，欲藉拮抗Pim1與下游蛋白的結合，減緩肝細胞癌的惡化情形，並為了讓胜肽更能有效被細胞吞噬，在其胜肽N端接上細胞穿透胜肽。在本篇論文中，我們首先藉由免疫螢光染色確定Pim1及RBMY皆表現在細胞粒線體位置，並利用螢光胜肽證實當RBMY胜肽被肝癌細胞吞噬後同樣會進入到粒線體胞器中。接著為了測試RBMY胜肽在不同細胞中的抑制效果，我們利用三種不同的肝癌細胞PLC/PRF/5、Huh-7及SNU423，先透過西方點墨法了解細胞本身Pim1及RBMY蛋白的表現差異，並將RBMY胜肽加入不同細胞後，探討三段RBMY胜肽在細胞的抑制效果。首先我同樣利用西方點墨法確認Pim1下游受質磷酸化受到抑制的蛋白，並利用相關的細胞功能試驗分別測試細胞的爬行、穿透及幹性能力。我們發現在三種細胞中SR08最能抑制細胞形成3D團塊，而在細胞爬行試驗中，SR08及SG08分別可減緩細胞爬行能力。此外，YR08及SR08皆可抑制細胞穿透，顯示RBMY胜肽可能具有作為肝癌之胜肽抑制劑的潛力，未來可將YR08、SR08及SG08三種胜肽協同使用於肝癌細胞中，藉由同時抑制細胞多條路徑而使胜肽抑制劑的抑制效果加成。

Hepatocellular carcinoma (HCC) is one of the leading cancers with high incidence rate in male and high mortality in the world. The first approved drug for HCC is Sorafenib, which causes severe side effects and shows limited survival improvement. Our previous studies showed that RNA Binding-Motif on Y chromosome (RBMY) reactivated and highly phosphorylated in male HCC tissues. RBMY protein is characterized by an RNA recognition motif (RRM) and C-terminal with four repetitive segments rich in serine, arginine, glycine and tyrosine residues (SRGY boxes), which is identified as intrinsically disordered proteins and generally considered difficult for developing drugs. Though intrinsically disordered sequences lack of constant protein structure, its flexibility make it easily target with multiple proteins and regulate cell functions. We therefore targeting the upstream kinase of RBMY, Proviral Integration in Moloney-1 (Pim1). To develop membrane-permeable peptide inhibitors which prevent the phosphorylation-activation of RBMY by Pim1, several candidate peptide sequences identified from RBMY sequence were synthesized with conjugated cell penetrating peptide (CPP) to their N-terminus terminus for higher endocytosis efficiency. In the study, we tested the efficacy of peptides inhibitors by different cell functional assays in three HCC cell lines. First, we confirmed that Pim1, RBMY proteins and our RBMY peptides were located at mitochondria. Besides, we treated peptide inhibitors in three HCC cell lines PLC/PRF/5, Huh-7 and SNU423. We used Western blot assay to investigate which Pim1 downstream proteins can be affected by RBMY peptides. Then, we used wound healing, transwell and spheroid formation assays to test cell migration, invasion and stemness abilities, respectively. The results showed that SR08 peptide could inhibit HCC cells to form spheroid cells. Furthermore, SR08 and SG08 peptides could slow down cell migration; YR08 and SR08 could inhibit cell invasion progression. In conclusion, our studies confirm that RBMY peptide inhibitors have potential prospects in HCC treatment. In the future, three peptide inhibitors could combined in HCC cell lines for better effect by inhibiting multiple pathways.