視黃酸誘導性基因-I (RIG-I) 透過DKK1抑制肝細胞癌的癌幹性以及Wnt訊息傳遞

Retinoic Acid-inducible Gene I( RIG-I ) Suppresses Stemness of Hepatocellular Carcinoma and Wnt Signaling by Regulation DKK1

褚名尉(Ming-Wei Chu)

指導教授 ： 華國泰

國立臺灣大學/醫學院/毒理學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201610173

摘要

肝細胞癌 (Hepatocellular carcinoma ; HCC) 是全球好發率排名第五位的癌症，由於治療後肝癌復發的比率高居不下，手術後五年存活率僅介於35%及65%之間。其中有 80-90 % 的肝癌是由於B型、C型肝炎病毒的感染導致。癌症幹細胞 (Cancer stem cell) 是一群具有自我更新、增殖與分化特性的細胞，並具有球體形成 (sphere formation) 能力。近年研究也證實癌症幹細胞與癌症的復發、轉移以及對化療藥物的抗藥性有極大的關聯性。視黃酸誘導基因-1 (Retinoic acid-inducible gene I; RIG-I) 是體內先天性免疫系統的重要成員，當RIG-I偵測到外來RNA病毒例如C型肝炎病毒入侵後，會活化mitochondrial antiviral-signaling protein (MAVS)，同時促使nuclear factor kappa B (NF-κB) 等免疫相關轉錄因子活化，最終產生第一型干擾素及前發炎性細胞激素。在過去許多研究指出 RIG-I 在肝癌細胞中表現量較其正常細胞中低;而在沒有外來病毒感染的情況下， RIG-I 的表現已知會影響胚胎發育及細胞分化的過程。另一方面， RIG-I 已發現會抑制間質幹細胞 (Mesenchymal stem cell) 及造血幹細胞 (hematopoietic stem/progenitor cells) 的生長，在老鼠的胚胎幹細胞中也有 RIG-I 表現下降的情況。然而 RIG-I 在肝癌幹細胞中扮演的角色仍不清楚，因此本研究欲探討 RIG-I 在肝癌中參與的生物功能及其調控癌症幹細胞機轉。首先在細胞實驗我們發現sphere cell和貼附細胞相比 RIG-I 的表現量較低且癌症幹細胞標記 Oct4和 OV6 表現量較高，透過人工合成的雙股RNA poly I:C活化 RIG-I 後能降低sphere的數量及大小，而抑制 RIG-I的表現後則會使肝癌幹細胞的標記蛋白表現量上升，同時伴隨 sphere 增加的情況。在細胞株轉染 RIG-I 不同片段的實驗中發現，凋亡蛋白酶募集區域 (capase recruitment domain, CARD domain) 的表現會抑制 sphere 生長。另外，我們發現在抑制 RIG-I 表現後觀察到β-catenin 有入核的現象，顯示RIG-I 可能透過活化 (Wnt signaling pathways) 促使調控細胞生長之基因例如c-myc與cyclin D1過度表現，進而造成細胞轉化成癌細胞 (cell transformation) ，甚至趨使已分化的細胞轉變成癌症幹細胞。在動物實驗中也證實RIG-I的減少會增加腫瘤的發生率，而同時再抑制β-catenin則會使腫瘤發生率再次降低。最後我們進一步RIG-I可能透過NFkB1及DKK1來調控Wnt訊息傳遞路徑。綜合以上觀察，我們希望能證實 RIG-I 在肝癌中抑癌基因及調控癌症幹細胞的角色，並作為未來研究肝癌治療的新方向。

關鍵字

視黃酸誘導性基因-I ； 肝細胞癌 ； 癌幹性 ； Wnt訊息傳遞

並列摘要

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third most common cause of cancer mortality. However, high rate of recurrence remains the major cause of death among HCC patients. The 5-year survival rate of HCC after treatment is only 35% to 65%. Approximate 80-90% of HCC patients are HBV or HCV carriers in the world. Cancer stem cells (CSCs) are small population of cells within a tumor. These cells possess the self-renewal ability, sphere formation ability, multilineage differentiation potential, and the potential to proliferate extensively. Recent studies have found cancer stem cells are associates to drug resistant, metastasis and tumor recurrence after therapies. Retinoic acid-inducible gene I (RIG-I), also named DDX58, is an important component of the innate immune response. When the virus, such as HBV or HCV infect host cells, RIG-I may act as a sensor then activates MAVS (mitochondrial antiviral signaling protein) and trigger an antiviral response by inducing interferon-β (IFN-β) production. In the previous studies, RIG-I reduction was found in liver cancer cells compared with normal cells. RIG-I also affects embryo development and cell differentiation without virus infection. On the other hand, the expression of RIG-I may reduce the proliferation rate of mesenchymal stem cell and hematopoietic stem/progenitor cell proliferation. The expression of RIG-I also decreases in mouse embryonic stem cells. However, the role of RIG-I in liver cancer stem cell is still unknown. Accordingly, the aim of our study was to evaluate the role of RIG-I in HCC and the mechanism leading to malignant transformation of liver cancer stem cells. First, we found that sphere cells have lower expression of RIG-I and higher expression of cancer stem cell markers Oct4 and OV6 than adherent cells. Activating RIG-I by poly (I: C) transfection could reduce the number and size of sphere cells. Furthermore, knockdown of RIG-I increase the liver cancer stem cell markers and the number of spheres. After transfection with different domains of RIG-I, RIG-I-full-length (wild type), RIG-I-N (constitutively active), RIG-I-C (dominant negative) constructs, we found that both full-length and N-terminal could inhibit the ability of sphere formation. In addition, we found that knockdown of RIG-I led to a nuclear translocation of β-catenin. This result implied that RIG-I might regulate cancer cell stemness through Wnt signaling pathways. In the in vivo experiment we found RIG-I deficiency increased tumor incidence. Meanwhile, knockdown β-catenin would reduce tumor incidence again. Finally, we further found RIG-I might regulate Wnt signaling through NFkB1 and DKK1. Taken together, we have shown the importance of RIG-I in the initiation and development of HCC and targeting RIG-I may have potential as a therapeutic approach to treat HCC.

並列關鍵字

RIG-I ； Hepatocellular carcinoma (HCC) ； stemness ； Wnt signaling

參考文獻

Alves, R., Alves, D., Guz, B., Matos, C., Viana, M., Harriz, M., Terrabuio, D., Kondo, M., Gampel, O., and Polletti, P. (2011). Advanced hepatocellular carcinoma. Review of targeted molecular drugs. Ann Hepatol 10, 21-27.

Ban, K.C., Singh, H., Krishnan, R., and Seow, H.F. (2003). GSK-3β phosphorylation and alteration of β-catenin in hepatocellular carcinoma. Cancer letters 199, 201-208.

Chang, M.H. (2003). Decreasing incidence of hepatocellular carcinoma among children following universal hepatitis B immunization. Liver international 23, 309-314.

de La Coste, A., Romagnolo, B., Billuart, P., Renard, C.-A., Buendia, M.-A., Soubrane, O., Fabre, M., Chelly, J., Beldjord, C., and Kahn, A. (1998). Somatic mutations of the β-catenin gene are frequent in mouse and human hepatocellular carcinomas. Proceedings of the National Academy of Sciences 95, 8847-8851.

El-Serag, H.B. (2011). Hepatocellular Carcinoma. New England Journal of Medicine 365, 1118-1127.