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Title: | 利用核醣核酸干擾篩選技術探討肝癌模式中對Sorafenib具感受性之基因 Identification of sorafenib-sensitizing genes in hepatocellular carcinoma by RNAi screening |
Authors: | Sheng-Wei Tseng 曾聖為 |
Advisor: | 周綠蘋(Lu-Ping Chow) |
Keyword: | 肝細胞癌,核醣核酸干擾篩選技術,蕾莎瓦,脾酪胺酸激酶, Hepatocellular carcinoma,RNA interference screening,sorafenib,Syk, |
Publication Year : | 2013 |
Degree: | 碩士 |
Abstract: | 肝癌在人類社會中已成為盛行率及死亡率高的癌症之一,在國內亦是危害國人健康的頭號殺手。然而,由於我們對於造成肝癌的因子,包含相關基因、蛋白質分子、細胞狀況甚至環境的機轉及交互作用仍缺乏全面性的了解,導致其治療困難且預後不佳。而目前經FDA核准的唯一一個肝癌標靶治療藥物是蕾莎(sorafenib),為一個多種激酶(kinase)的抑制劑,可抑制肝癌細胞生長並促進細胞死亡;然而,其療效仍然有限,因此探討其可能影響的未知分子標的已成為肝癌治療上重要的研究課題之一。
近年來,利用核醣核酸干擾技術探討基因間的交互作用,以及鑑定對藥物具感受性的基因已被廣泛應用,在此基礎上,我們希望能建立在異種移植動物模式(xenograft model)中進行核醣核酸干擾篩選的平台。因此,初步我們挑選了96種激酶(kinase),並以小髮夾RNA(shRNA)庫抑制其基因表現,比較在小鼠中經藥物處理前後的腫瘤其shRNA對於基因抑制的變化情形。 經次世代定序(nexe-generation sequencing)與統計分析後,發現3個基因的shRNA讀值在經藥物處理前後有顯著改變,而其中脾酪胺酸激酶(Syk)於先前的研究被指出在某些癌症中是具潛力的藥物標靶,因此我們挑選了Syk進行後續實驗。 我們首先利用shRNA與抑制劑分別抑制HuH-7細胞中的Syk表現並以西方墨點法確認抑制效果。之後利用細胞生存試驗比較正常細胞與Syk受抑制的細胞,發現Syk被抑制後細胞對sorafenib的感受性增加,50%抑制濃度(IC50)由6.7μM下降至4.0μM。另外我們也發現,和正常的HuH-7細胞相比,單純抑制Syk並不會對細胞生長造成影響,但若同時加入sorafenib,則會使細胞生長受明顯抑制,雖然其中的詳細機制及Syk在肝癌中可能扮演的角色、功能仍須進一步探討,但期望此平台能應用於更大規模的篩選及其他疾病的研究中。 Hepatocellular carcinoma (HCC) is one of the most lethal and prevalent cancers in humans. Despite its significance, there are only limited numbers of effective therapeutic options, partially because our understanding of the genetic, molecular, cellular and environmental mechanisms that drive disease pathogenesis is far from comprehensive. So far, the only one molecular targeted therapy drug for HCC is sorafenib, which is a multi-kinase inhibitor. However, the mechanism underlying the therapeutic effect of sorafenib remains unclear, and several “off-targets” were recently discovered. Thus, to identify targets that sensitize to sorafenib may provide important information on HCC treatment. Recently, genome-wide target gene knocked down by the pooled RNAi Consortium (TRC) shRNA library has been widely used, and it can be combined with drug treatment to become a platform for identifying potential drugs with synergistic effect. Based on this technology, we want to identify the sorafenib-sensitizing targets in HCC by in vivo RNAi screening. In our study, we transfected the pooled shRNA packaged by lentivirus into a HCC cell line, HuH-7 and injected these resulting cells subcutaneously into nude mice to knock down 96 kinases. After tumors grew, the nude mice were then divided into two groups, control and sorafenib treatment. After analyzing the genomic DNA of tumors by next-generation sequencing, three genes were found that their shRNA ratios were dramatically different between treatment and control groups. Although the functional roles of these genes among the list in resistance to sorafenib are still undetermined, Syk has been reported as a potent modulator of epithelial cell growth and may be a potent molecular target in cancers. We then used shRNA and its specific inhibitor, BAY 61-3606, to knock down Syk activity in HuH-7 cells. By cell viability assay, the HuH-7 cells are more sensitive to sorafenib, and the IC50 of sorafenib drops from 6.7μM to 4.0μM as Syk is inactivated. Furthermore, the growth rate of Syk silenced HuH-7 cells was also much slower than that of HuH-7 control cells, which suggested that Syk may have a synergistic effect with sorafenib on cell growth or survival and may be a potential target in HCC treatment. Although the regulation mechanisms and functions of Syk still need to be further elucidated, the screening platform in this study can provide us some potential targets on disease treatment. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/5980 |
Fulltext Rights: | 同意授權(全球公開) |
Appears in Collections: | 生物化學暨分子生物學科研究所 |
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