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標題: | 以蛋白質體學方法鑑定蕾莎瓦治療人類肝細胞癌後之預後生物標記 Identification of Potential Prognostic Biomarkers for Sorafenib Efficacy in Hepatocellular Carcinoma Cells by Proteomic Approaches |
作者: | Ming Jiun Tsai 蔡銘駿 |
指導教授: | 周綠蘋(Lu-Ping Chow) |
關鍵字: | 肝細胞癌,生物標記,分泌蛋白體學,CTGF,Galectin-3,Glypican-3,HMGB1, Hepatocellular carcinoma,biomarker,secreted protein,CTGF,Galectin-3,Glypican-3,HMGB1, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 肝癌是世界上最常見的癌症之一。其中有百分之七十五的肝癌屬於肝細胞癌 (hepatocellular carcinoma, HCC)。在臺灣,肝癌的盛行率為所有癌症的第二名,僅次於肺癌。目前臨床上已有許多生物標記可用來診斷與評估肝癌治療後的預後,也有許多具潛力的生物標記正在被研究。根據 Barcelona-clinic liver cancer staging (BCLC staging),晚期的肝癌病人常使用唯一的口服肝癌標靶藥物 sorafenib 治療。Sorafenib 是一種多重激酶抑制劑,能藉由 Raf 抑制 MAPK 細胞傳遞路徑,且是目前唯一美國 FDA 核可上市的口服肝癌標靶藥物,再加上其副作用相對和緩而被用來治療肝細胞癌後期之病人。然而,目前並沒有一個好的方法能用以預測 sorafenib 治療肝癌病人後的預後。近來雖然有許多的研究著力於 sorafenib 治療效果相關生物標記的探討,並沒有找到適當的生物標記能應用到臨床上以評估預後。因此找到能用以預測肝癌在 sorafenib 治療後之療效之生物標記是刻不容緩。
以定量蛋白質體學 (quantitative proteomics) 的方法,細胞培養中標記穩定同位素胺基酸 SILAC (stable isotope labeling of amino acids in culture) 之技術,分析HuH-7 在 sorafenib 治療前與治療後,細胞內及分泌性蛋白質表現量的差異,並以質譜儀 LC MS/MS 鑑定這些蛋白質,再以生物資訊分析軟體IPA (ingenuity pathway analysis) 對這些胞內與分泌性的蛋白質做疾病與功能上的分析。分別在細胞裂解液與細胞培養液中鑑定到2611個與1022個蛋白質,且分別有1091個與145個蛋白質的表現量是下降的。再將這些蛋白質以生物資訊分析軟體 TMHMM、SecretomeP 以及 SignalP 分析,發現在細胞裂解液與細胞培養液中共有103個蛋白質屬於分泌性蛋白質。這些蛋白質皆與細胞凋亡、細胞增生、血管生成與癌症有關,並從中篩選出四個有潛力之生物標記 CTGF、Galectin-3、Glypican-3 及 HMGB1。再分別以活體外之細胞實驗及活體內小鼠模式動物進行驗證,這四個蛋白質的表現量都會因sorafenib的治療而下降,且在活體內的腫瘤生長也會因sorafenib 的治療而受到明顯抑制。並也探討這些蛋白質所參與的細胞傳遞路徑,發現這些蛋白質皆屬於 MAPK/ERK 下游之表現蛋白質。另外也觀察細胞的生長情形,將蛋白質GPC3的表現抑制後,細胞的生長也會受到抑制。根據我們的研究結果,我們找到四個具有潛力的生物標記可以用來預測肝癌病人經 sorafenib 治療後之預後狀況。這些生物標記需更多臨床上相關的研究及驗證,從而了解其價值,並應用到臨床。 Hepatocellular carcinoma, HCC, is the most common liver cancer in the world. Clinically, there are many biomarkers which can be used to diagnose HCC and predict prognosis. According to the Barcelona-clinic liver cancer staging (BCLC staging), sorafenib is the only anticancer drug with proven prognostic efficacy for treatment of HCC. Sorafenib is a multi-kinase inhibitor and inhibits the MAPK pathway by inhibiting Raf. Because sorafenib is the only approved drug for advanced HCC and exhibits relatively mild adverse effect, biomarkers which can be used to predict sorafenib efficacy can assist in identifying the patients who are likely to benefit from the treatment. Many studies have attempted to investigate biomarkers of sorafenib efficacy by analyzing the associations between potential markers and patients’ outcomes. However, there are no appropriate biomarkers can be used to estimate prognosis. Therefore, it is necessary to find biomarkers which can be used to predict the prognosis of HCC patients after sorafenib treatment. We applied the quantitative proteomics method (Stable Isotope Labeling of Amino acids in Culture, SILAC) to analyze the differences of secretory and cytosolic proteins levels between HuH-7 and sorafenib-treated HuH-7 cells. We further used the LC MS/MS approach to identify the above proteins. The Ingenuity Pathway Analysis (IPA) was performed to analyze the difference of functions between secretory and cytosolic proteins of HuH-7 and sorafenib-treated HuH-7 cells. According to our results, we identified 2611 proteins in cell lysate and 1022 proteins in conditioned medium of sorafenib-treated HuH-7 cells. Of these proteins, there are 1091 and 145 proteins down-regulated in cell lysate and conditioned medium respectively after sorafenib treatment. Furthermore, we analyzed these two groups by bioinformatics software, TMHMM, SecretomeP and SignalP, and totally 103 proteins were classified as the secretory proteins. These proteins were found to be relating to apoptosis, proliferation, angiogenesis and cancer. In which, we selected four candidates, CTGF, Galectin-3, Glypican-3 and HMGB1, which may be potential biomarkers for predicting prognosis of HCC patients after sorafenib treatment. We validated these potential biomarkers by western blot in vitro and immunohistochemistry in vivo and found that the expressions of these four proteins are inhibited by sorafenib. In addition, the tumor volume was also decreased in vivo after sorafenib treatment. We further studied the involved cell signaling of these potential biomarkers with several inhibitors and found that these four proteins are regulated by MAPK/ERK pathway. Moreover, proliferation was also inhibited in GPC3-knowdown HuH-7 cells. In conclusion, we found that these four proteins are potential biomarkers for predicting prognosis of HCC patients after sorafenib treatment. It is expected to make these potential biomarkers be valuable in clinical use. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68757 |
DOI: | 10.6342/NTU201703750 |
全文授權: | 有償授權 |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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