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標題: | 肝細胞癌中Robo1訊息傳導功能之研究 Functional Study of Robol Pathway in Hepatocellular carcinoma |
作者: | Han-Hsiang Huang 黃涵湘 |
指導教授: | 許輝吉 |
關鍵字: | 肝細胞癌, Robo1,hepatocellular carcinoma, |
出版年 : | 2006 |
學位: | 碩士 |
摘要: | ROBO1 (Roundabout human homologous-1) 是一位於細胞膜上的接受器,最初發現在果蠅的神經發育過程中,和其配位子 (ligand) SLIT2鍵結後,會產生排拒作用,而使神經細胞不往有SLIT2的方向移動。ROBO1和SLIT2造成的排拒作用,也出現在其他物種,其他種類的細胞和癌細胞中,如:白血球細胞,乳癌細胞。近年來的研究發現,ROBO1在肝細胞癌及直腸癌,有過量的表現。本實驗室利用mRNA分示法,發現ROBO1 variant 2 isoform (ROBO1B) 在肝細胞癌過量表現。
因為發現ROBO1B在肝細胞癌中過量表現,因此我們利用RNAi技術,降低ROBO1B的表現量,來探討ROBO1B在肝細胞癌中扮演的角色 (oncogene or tumor suppressor gene?)。實驗結果發現,ROBO1B表現量降低後。HA22T細胞株在細胞移動實驗及transwell實驗中,細胞移動及侵犯的能力皆下降 (p value分別是:p<0.01, p<0.01),另外HA22T及HuH-7兩株細胞株在3D collagen I gel 實驗中,ROBO1B表現量降低的細胞,生長出比控制組少且短的突起。在細胞生長能力方面,HA22T細胞株ROBO1B表現量降低後,在細胞生長 (MTT assay) 和細胞非貼附性生長 (soft agar assay) 實驗,生長速率都增加 (p<0.05, p<0.001)。但是相反的,ROBO1B表現量降低後,在HuH-7細胞株,細胞生長並不受影響,而細胞非貼附性生長 (soft agar assay) 實驗,生長速率卻是降低 (p<0.005),在以HuH-7建立的穩定細胞株B4, B14, B43中,在細胞非貼附性生長 (soft agar assay) 實驗,和In vivo.的動物實驗,生長速率皆降低 (分別soft agar:p<0.0001, p<0.0001, p<0.005,動物實驗:p<0.01, p=0.02, p=0.17)。在細胞貼附實驗上, HuH-7細胞株對collagen IV及laminin的貼附力降低 (p<0.005, p<0.01),但HA22T細胞株對vitronectin的貼附力卻升高 (p<0.001)。我們尋找這兩株細胞在ROBO1B的表現量降低後,細胞生長和細胞貼附實驗呈現不同表現的原因。 我們檢測細胞內與ROBO1B關係密切的SLIT2之表現量,發現HA22T ROBO1B表現量降低後,SLIT2表現量升高,HuH-7則是無SLIT2的表現。在我們做了臨床病理學的分析後發現,若將ROBO1B和SLIT2的表現量做交叉比對,發現對細胞最好的情況是ROBO1B和SLIT2都高量表現,ROBO1B或SLIT2任一方表現量降低時,腫瘤惡化的程度都明顯升高,因此推論,內生性ROBO1B和SLIT2表現量的平衡,是非常重要的。因此我們推論,在討論ROBO1B對細胞造成的影響的同時,或許不能忽略SLIT2的重要性,探討ROBO1B和SLIT2在細胞中的表現,甚至和其他相關分子之間相互影響,是我們將來的課題。 另外在細胞培養的過程中,意外的發現HA22T及HuH-7兩株肝癌的細胞株,ROBO1B表現量會隨著細胞密度增加而升高。將細胞施予抑制劑後,兩株細胞在有curcumin抑制劑 (AP-1抑制劑—JNK訊息傳遞路徑之轉錄因子) 存在下,ROBO1B表現量皆降低。收取培養第二天及第五天的細胞,測量蛋白表現量,發現細胞在第五天時,ROBO1B和c-jun (AP-1 subunit) 的表現皆升高。已知ROBO1B有兩個AP-1的鍵結位置 (binding site)。因此我們推論,ROBO1B可能被JNK訊息傳遞路徑所調控。 ROBO1 (Roundabout human homologous-1), a transmembrane receptor, was first discovered in Drosophila neuron development. Interaction with its ligand - SLIT2, ROBO1 causes a repellent signaling preventing the cell migration toward SLIT2 side. The repulsion effect also occurs in other cell types or tumor cells in different species, such as human leukocytes and breast cancer. Recent studies have shown the overexpression of ROBO1 in human hepatocellular carcinoma (HCC) and colorectal cancer. We also observed that ROBO1 variant 2 (ROBO1B) was often overexpressed HCC. With its frequent overexpression in HCC and its membrane protein nature, we tried to evaluate the potential of ROBO1B as a novel therapeutic target of HCC using the RNAi technique to knockdown ROBO1B. We demonstrated that the ROBO1B knockdown could significantly block the cell migration in wound healing and transwell invasion capability of HA22T (p<0.01, p<0.01). Further, ROBO1B knockdown could dramatically reduced the invasion phenotype of both HA22T and HuH-7 HCC cells in 3-D gel, as manifested by the fewer and shorter cytoplasmic processes. Despite the similarities of the reduced invasive phenotypes, HA22T and HuH-7 cells exhibited significant differences in cell growth ability after RNAi knockdown of ROBO1B in both anchorage-dependent and –independent cell growth. ROBO1B RNAi by transient transfection of RNAi oligos led to dramatic decrease in the colony formation ability in soft agar assay, p<0.005, in HuH-7 cell line. Further, we also established three stable lines of ROBO1B knowndown HuH-7 cells (named B4, B14, B43), which also showed significant decrease in anchorage-independent growth, p<0.0001, p<0.0001, and p<0.005, respectively. These three stable knockdown clones also exhibited reduction in tumor growth in SCID mice, p<0.01, p=0.02, and p=0.17, respectively. In contrast to our expectation, ROBO1B knockdown caused increased in cell growth of HA22T cells in both MTT and soft agar assay (p<0.05 and p<0.001, respectively). We then did cell adhesion assay. ROBO1B knockdown HuH-7 cells had decreased adhesion ability to collagen IV and laminin (p<0.005, p<0.01), whereas ROBO1B knockdown HA22T cells showed increase in adhesion ability to vitronectin (p<0.001). Besides, we found that SLIT2 mRNA level remained undetectable in HuH-7 cells, whereas the SLIT2 levels increased in HA22T cells after ROBO1B knockdown. These contradictory findings, together with the close relation between ROBO1B and SLIT2 in axonal guidance, and the opposite expression, ROBO1B overexpression and SLIT2 downregulation in HCC, prompted us to review the role of ROBO1B and SLIT2 in human HCC. We found that HCC with expression of both ROBO1B and SLIT2 had lower frequency of vascular invasion as compared with HCC with ROBO1B overexpression but SLIT2 downregulation, p=0.028, suggesting that SLIT2 is an endogenous inhibitor of ROBO1B-mediated tumor invasion. More studies are warranted to elucidate the complex interacting roles of ROBO1B and its ligands in HCC and very likely other types of human cancer. In the study of cell growth, it came to our surprise that the mRNA levels of ROBO1B of both HA22T and HuH-7 cells were increased as the cell density increased. Using specific inhibitors, both cell lines exhibited dramatic morphologic changes, manifested by cell round-up, and had dramatically decreased ROBO1B mRNA level after curcumin (AP-1 inhibitor - the transcription factor of JNK pathway) treatment. These findings suggest that ROBO1B is regulated by the AP-1 pathway, and deserves to be further elucidated. Consistent with this suggestion, ROBO1B has two AP-1 sites in the promoter region. Besides, ROBO1B and c-jun protein levels of HA22T and HuH-7 cells increased in parallel at high cell density (day 5 culture) as compared with culture at low cell density (day 2). These findings suggest that ROBO1B is regulated by JNK pathway. We also showed that ROBO1B in HA22T was decreased after LY294002 (PI3K inhibitor), suggesting that PI3K might be also involved in the activation of ROBO1B transcription. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/31491 |
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