探討組蛋白去甲基化酶PHF2在胃癌進程之角色

The Roles of Histone Demethylase PHF2 in Gastric Cancer Progression

毛采月(Tsai-Yueh Mao)

指導教授 ： 華國泰

國立臺灣大學/醫學院/毒理學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201800324

摘要

胃癌佔了國人十大死因的第七位，臨床上證實胃癌細胞的遠端轉移是造成胃癌病人存活率低的主要原因。故尋找能夠調控胃癌進程的專一性分子對於發展標靶治療藥物相當重要。組蛋白去甲基化酶(histone lysine demethylases, KDMs) 可移除組蛋白上的甲基，藉此可正向或負向調控DNA轉錄以及蛋白質功能，其中plant homeodomain finger protein 2 (PHF2)是組蛋白去甲基化酶之中含有Jumonji C (Jmj-C)特殊氨基酸序列的蛋白家族之一。在過去的文獻中曾報導PHF2在不同癌症中，可能透過不同的機制扮演致癌或是抑癌基因，本研究中我們試圖探討PHF2在胃癌中的角色，評估其做為新穎胃癌藥物標靶或生物標誌的可能性，首先經由分析臨床資料庫The Human Protein Atlas (THPA)以及The Cancer Genome Atlas (TCGA)，發現在PHF2蛋白在胃癌病人中有高度表現且表現量高的病人有較差的存活率，可做為預後指標分子。在細胞實驗中抑制PHF2在胃癌細胞株的表現量，觀察到細胞生長速度、遷移與入侵能力皆顯著下降。我們同時利用transwell篩選出具有較高度侵襲能力的細胞株AGS-H5，並發現相較於parental AGS細胞，AGS-H5有較高的PHF2表現。另外在in vivo實驗中使用NSG小鼠，以腹腔注射方式給予胃癌細胞AGS-H5建立胃癌腹腔轉移動物模式實驗，在第四週利用IVIS冷光觀察腸胃消化道及腸繫膜上有腫瘤生成，而抑制PHF2表現的組別則不見明顯的腫瘤。為了進一步釐清PHF2可能調控的基因，進行Affymetrix Microarray分析，結果發現在抑制PHF2表現後，lncRNA H19的表現也被抑制，在過去研究指出H19的高度表現會促進多種惡性腫瘤的進展，而我們也發現臨床上胃癌病人中的PHF2與H19表現量具有正相關性。綜合以上觀察，PHF2可能透過調控H19的表現來調控胃癌的進展。因此，PHF2在胃癌機制上的探討或許能成為有用的生物標記以及具有發展治療胃癌藥物的潛力。

關鍵字

去甲基化酶 ； 胃癌 ； 表觀遺傳

並列摘要

Gastric cancer (GC) ranks the 7th of the 10 leading cause of death in Taiwan in recent years, and the survival rate of advanced gastric cancer patients with distant metastasis is very low. It is urgent to find out a molecule that is specific to gastric cancer progression as a therapeutic target. The histone demethylase plant homeodomain finger protein 2 (PHF2) has an enzymatically active Jumonji-C domain (JmjC) and demethylated lysine residues of protein to regulate protein function. In previous studies, PHF2 plays multiple roles in regulating cancer progression. Here, we investigated the role of PHF2 in gastric cancer to evaluated its potential to be a novel therapeutic target. We analyzed The Human Protein Atlas (THPA) and The Cancer Genome Atlas (TCGA) databases and discovered that higher levels of PHF2 were observed in GC patients and correlated with poor prognosis. We hypothesized that PHF2 is an oncogene in GC. In vitro, we knocked down PHF2 in GC cells and found that cell proliferation, migration and invasion ability were significantly down-regulated. We also selected invasive sublines, AGS-H5, though transwell system and showed that AGS-H5 expressed higher PHF2 compared to parental AGS cells. In in vivo study, a peritoneal metastasis animal model was established by the intraperitoneal injection of AGS-H5 cells and observed tumor formation in mesentery by IVIS spectrum. We found that knocking down PHF2 inhibited tumor growth in vivo. On the other hand, the mRNA levels of whole genes affected by PHF2 were measured by Affymetrix Microarray in GC cells. The putative gastric tumor metastasis-associated genes, lncRNA (long non-coding RNA) H19, were identified. We confirmed that H19 expression was inhibited after PHF2 down-regulated and discovered that PHF2 expression level had positive correlated with H19 expression level. As mentioned above, our results demonstrated that PHF2 may involve in gastric cancer progression by regulating H19 expression. Further evaluation of the mechanisms of PHF2 in cancer may provide the novel therapeutic methods on anti-cancer drug development.

並列關鍵字

PHF2 ； gastric cancer ； demethylase

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