鈣離子訊號傳遞在HBx蛋白誘導事件中的重要性

Abstract

在全球，肝癌的致死率居高不下，其中在原發性肝癌中，以肝細胞癌(Hepatocellular carcinoma，HCC)佔了最高的比例，為75-85%，而慢性B型肝炎病毒(Hepatitis B virus，HBV)感染為肝細胞癌的主因。然而，慢性HBV感染導致肝細胞癌的原因仍是個未解的難題。在過去的研究中，由於B型肝炎病毒X蛋白(Hepatitis B virus X protein，HBx)與HCC有緊密的關聯，例如：在與HBV感染相關的HCC病人上可以偵測到HBx的表現，以及表現HBx的基因轉殖鼠有較高生成肝腫瘤的發生率，同時，HBx也曾被報導與HBV的複製及轉錄相關，因此HBx是科學家們研究肝細胞癌機制的標的之一。除此之外，HBx與鈣之間有所連結：HBx會與和B細胞淋巴瘤-2 (B-cell lymphoma 2，Bcl-2)，以及線蟲中與此蛋白同源的細胞死亡異常基因9 (Cell death abnormally gene 9，CED-9)蛋白進行交互作用來提升細胞內鈣離子的訊號，同時，鈣離子訊號的改變也會影響HBx所促進的HBV複製現象。由以上的發現，推測HBx或許會藉由改變鈣離子訊號傳遞，並進而導致肝癌。關於HBx對於鈣離子恆定的影響，由實驗室的其他同仁進行探討中，本篇研究將定睛於解答：鈣離子訊息傳遞是否在HBx所誘導的事件中，扮演了不可或缺的角色之問題。研究的策略為：(1)建立可信賴的平台闡述HBx所造成的影響；(2)嘗試找到操縱鈣離子傳遞訊號及在過程中精確地測量鈣濃度的方式，確保實驗結果與所建立實驗環境之對應；(3)皆完備後，嘗試回答研究目的。 本篇研究有兩個初步的發現：HBx使得細胞數目減少，以及HBx與上皮生長因子受體(Epidermal growth factor receptor，EGFR)之間可能有交互作用。我們發現使用慢病毒感染使得HepG2人類肝細胞癌肝上皮細胞表現HBx時，將使得細胞數量下降，並在這個發現的基礎上，嘗試使用Thapsigargin、EGTA等鈣離子試劑觀測其對於HBx組與控制組之間細胞數量的影響，我們觀察到：HBx對於Thapsigargin的IC50沒有影響，但當創造高鈣離子濃度的環境時，HBx似乎會導致細胞數量減少的程度下降。因此，我們計畫以更精確的平台，如Cell death assay和Cell cycle assay來觀測，期許可以獲得更清晰的結果。第二個初步的發現，則是由於在兩種細胞株，HepG2人類肝細胞癌肝上皮細胞與FL83B小鼠肝細胞上觀察到HBx似乎會降低EGF促使細胞增加的效果，使我們猜測HBx所造成的細胞數目下降可能依賴於EGFR信號通路，同時也因為EGFR的活化與鈣離子訊號傳遞之間有所關聯，因此進一步探討HBx對於EGFR之表現及其下游信號通路的影響，實驗的結果說明HBx所造成細胞數目的下降應與EGFR信號通路無關，而HBx會增強细胞外調節蛋白激酶(Extracellular regulated protein kinases，ERK)的磷酸化訊號，以及縮短蛋白激酶B (Protein kinase B，PKB /Akt)磷酸化訊號的持續時間。目前正在嘗試以GCaMP6s作為鈣指示劑，比較有無HBx是否會影響加入EGF之後鈣離子訊號的變化。 盼望在未來當平台的建立更加成熟，也找到適當的方式調控鈣離子訊號時，能精進現今的實驗結果，一步一步地朝著解答肝細胞癌機制之方向前進。

Hepatic cancer is one of the leading causes of cancer mortality globally. Hepatocellular carcinoma (HCC) accounts for 75-85% of primary hepatic cancer cases. Among various risk factors, the infection of hepatitis B virus (HBV) is the main cause of HCC. However, the mechanism of HBV-related HCC remains unknown. Previous studies have shown that an HBV-encoded protein: hepatitis B virus X protein (HBx), is tightly correlated with HCC because HBx expression was detected in most HBV-related HCC, and similarly, high incidences of liver tumors were found in transgenic mice expressing HBx. Besides, HBx was reported to regulate HBV replication and transcription, so it becomes a key to reveal possible resolutions to HCC. Additionally, it was found that Bcl-2 (and its homologue in Caenorhabditis elegans CED-9) was targeted by HBx to elevate intracellular calcium signaling. Furthermore, alteration of cytosolic calcium changed HBx-mediated HBV replication. From the above findings, we assumed that HBx may lead to HCC by regulating calcium signaling. The part of HBx influence on calcium signaling was investigated by our laboratory colleagues. Therefore, the aim of this study is to find out whether HBx-related effects are calcium-dependent. The strategy of this study includes three stages. (1) To build up reliable assays illustrating HBx effects. (2) To find out good ways to manipulate calcium signaling and calculate calcium concentration precisely during the experiment. (3) To observe the changes of HBx-induced effects influenced by calcium manipulation. Our two preliminary findings were that the cell count in HBx-infected group was decreased compared to the control one and there might be an interaction between HBx and epidermal growth factor receptor (EGFR) signaling. To evaluate HBx-induced cell number reduction, we used calcium reagents, such as Thapsigargin and EGTA, to observe their effect on the cell count of HBx and control group. We found that HBx has no impact on IC50 of Thapsigargin, but it seemed that HBx caused smaller extent of cell number reduction under higher calcium concentration. Thus, we will recruit cell death and cell cycle assay to further elucidate its underlying mechanisms. The second preliminary finding was that HBx seemed to reduce the effect of EGF-related cell number addition on two cell lines, HepG2 and FL83B. We thus proposed that HBx-induced cell number reduction might rely on EGFR signaling. Toghther with the known association between EGFR signaling and calcium activation, we decided to explore HBx effect on EGFR expression and its downstream signaling pathways. The result revealed that the cell count decrease caused by HBx was unrelated to EGFR signaling and that HBx enhanced the signal of phosphorylated ERK and shortened the duration of AKT activation. Currently, we are using GCaMP6s as a calcium indicator to compare the calcium signaling between HBx and control group after EGF treatment. In the future, we will keep optimizing above HBx-related assays and calcium measurement to reach a deep clarification of HBx-induced events and their variation influenced by calcium signaling alteration, so the goal to uncover the mechanism of HBV-related HCC could be achieved very soon.