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研究生:謝清源
研究生(外文):Chin-Yuan Hsieh
論文名稱:癌細胞中Robo1訊息傳導之功能研究
論文名稱(外文):Functional Study of Robo1 pathway in Cancer
指導教授:許輝吉許輝吉引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:病理學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
論文頁數:66
中文關鍵詞:肝癌癌症Robo1
外文關鍵詞:therapydifferentuationcancerRobo1
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中文摘要:
肝癌(HCC)是目前世上發生率以及致死率都極高的惡性腫瘤之一。肝癌的發生與肝損傷的關係密不可分,肝癌往往都伴隨著長期的發炎以及損傷而發生。 此外,在腫瘤發生時都會產生染色體的變異,如染色體的缺失,被高度甲基化,以及增幅(amplification)等等這些變化會導致基因表現的異常,我們應用differential display的方法來找出這些在腫瘤細胞和正常細胞之間表現有差異的基因。
Robo1正是我們找到會過量表現在HCC的基因之一,Robo1 (Roundabout human homologous-1)是一個位於細胞模上的蛋白接受器,它的mRNA長約8 Kb, 含有29個exon,分佈於240kb的染色體上,共可轉譯出1612個胺基酸,首先被發現在果蠅的神經發育中扮演著嚮導的角色。Robo1會與其細胞外的ligand,Slit2,共同作用,避免神經突處在發育時跨過體軸中線後形成紊亂的迴旋。 Robo1在神經細胞中接受了Slit2的訊息之後會活化下游的sr-GAP(GTPase active protein)蛋白,sr-GAP再去與CDC42競爭GTP的磷酸根,CDC42一旦失去磷酸根便失去活性,即無法驅使細胞內骨骼的聚合,Robo1藉此作方向性的抑制以驅使神經突處向既定的方向生長。除此之外,在近來的研究指出,Robo1的5’端的promoter在乳癌以及腎細胞癌中會被高度甲基化,因此Robo1被預期可能是一個抑癌基因。
然而我們發現一個被預期的抑癌基因會卻在HCC有過量的表現,同時我們查閱NCBI網站所公佈的序列,發現Robo1有2種異構物(Robo1A,Robo1B),於是我們利用RT-PCR的方法來觀察這兩個異構物的表現:
1. Robo1A只在胎兒的腦以及睪丸組織中被偵測到,而在成人組織中則偵測不到;而Robo1B會普遍在胎兒以及成人的組織中表現而在成人組織中的表現量要比在胎兒中的表現量大幅減少。
2. 在HCC中偵測不到Robo1A,而Robo1B會在59%的HCC病人的檢體中過量表現,但Robo1B過量表現的狀況與許多肝癌進行的風險因子沒有明顯的關聯。
3. 在利用retinoic acid誘發神經細胞(NT2)分化的實驗過程中,我們觀察到Robo1B的表現量會隨著分化的進程而減少。
4. 利用蛋白抑制劑(ZD1839,PD98059)處理Hep3B細胞的結果,Robo1B的表現量會在PD98059 (MEK1抑制劑)處理的Hep3B細胞中減少。
根據上述的觀察,Robo1A可能有對胎兒腦部的專一性表現;Robo1B的表現則有普遍性存在一般組織中,兩者都會在分化的過程中表現量降低。此外Robo1B在HCC中會過量表現,而且普遍過量表現在各種腫瘤的分期與級別中。表示Robo1B的表現可能會受MAPK的訊息傳遞影響。
我們在HeLa細胞中利用siRNA knockdown Robo1B的表現量以分析Robo1B可能的功能,在挑選了3株Robo1B siRNA穩定表現的HeLa 細胞後,觀察HeLa細胞在Robo1B1被knockdown後的改變: 細胞的外型會變大,細胞質會變多,核/質的比例會變小。細胞間的接觸會變得很緊密,F-actin的表現的更細密的佈滿整個細胞,並堆積在細胞的邊緣。分化的標籤基因(Keratin-10)表現量會增加, 生長速率會下降,侵犯與移動的能力都會降低。
HeLa細胞原本是分化不良的惡性腫瘤細胞株。在Robo1Bknockdown之後,細胞的形態變得比較接近已分化的角質細胞。而且其腫瘤的特徵也隨著Robo1B被knockdown減少。Robo1B是否可能成為有潛力的基因治療的目標,值得再研究。
Abstract
Hepatocellular carcinoma (HCC) is one of the most common fatal malignant neoplasms worldwide, and the primary cause of cancer-related death in some regions, particularly the South-east Asia, including Taiwan. Many genetic alternations have been reported in cancer, such as chromosome loss, promoter hypermethylation, and amplification etc. We used the differential display for a genome-wide screen to identify differentially expressed genes in HCC, frequentily.
Robo1 is one of the genes overexpressed in HCC. The Robo mRNA is 8 Kb long, with 29 exons spanning at least 24 Kb of the genomic sequence and encodes a big protein of 1612 amino acid residues. The roundabout (robo) gene was identified in a large scale Drosophila mutant screen for the genes that guide midline crossing of axons. Robo1 responds to its extracellular ligand, Slit2, and actives its downstream molecule, sr-GAP (GTPase active protein). Then sr-GAP competes the phosphate with CDC42. CDC42 is inactivated with the dephosphorylation, and can not trigger the actin cytoskeleton polymerization. Hence, the Robo1-Slit2 signal guides axon growth to the expected direction.
The promoter region of Robo1 has been shoen to be hypermethylated in breast cancer and renal cell carcinoma, and Robo1 may be a candidate tumor suppressor gene. However, Robo1 was often overexpressed in HCC using differential display. Here, we showed that Robo1 had two transcription isoforms (Robo1A and Robo1B). By specific RT-PCR using specific primers, we found:
1. Robo1A was predominantly expressed in fetal brain and testis, but undetectable in most adult tissues. Robo1B was expressed in the fetal and the adult tissues, but in much lower levels in the adult tissues.
2. Robo1A was undetectable in HCCs, but Robo1B was overexpressed in 95 out of 160 (59%) HCCs. The overexpression of Robo1B, however, did not significant association with the pathological risk factors for tumor progression.
3. In the retinoic acid induced neuronal differentiation of NT2 terato-carcinoma cell, the Robo1B expression was downregulated.
4. The Robo1B mRNA level was downregulated in the Hep3B cells is treated with PD98059 (MEK1 inhibitor), but not in cells treated with ZD1839 (EGFR inhibitor)
These observations suggest that Robo1A specificslly expressed in the fetal brain and testis, and Robo1B was developmentally downregulated and the downregulation was a feature of differentiation. Our results also suggest that the Robo1B expression may be regulated by MAPK signal pathway.
To elucidate the possible function of Robo1B, we used siRNA knockdown Robo1B in HeLa cells and obtained 3 Robo1B siRNA stable HeLa cells. We observed the following results: cells were enlarged with abundant cytoplasm with decreased N/C ratio. The cell-cell contact became much more distinct, and the F-actin fibers were coarse and distributed throughout the whole cytoplasm with condensation at the cell periphery at the cell-cell contact. The expression of the epithelial cell differentiation marker (Keratin-10) was increased. The growth rate, motility, and invasion ability decreased.
The parental HeLa cell is a malignant cell line. When we used siRNA knockdown Robo1B in HeLa cells, the morphology of the Robo1B knockdown HeLa cells were close to the differentiated keratinocytes. Besides, the malignant characters, as the growth rate, motility, and invasion ability, of HeLa cells are decreased with Robo1B knockdown. Whether Robo1B can be a potential therapeutic target must be worthy to further investigate.
目錄
目錄 i
中文摘要 1
Abstract 3
Introduction 5
1. HCC 6
(1) HCC 6
(2) The molecular changes in the HCC 7
(3) Genome-wide screening for genetic alterations 7
2. Robo1 9
(1) Discovery of ROBO1 gene 9
(2) The ligand of Robo: Slit 10
(3) The intracellular signal of Robo1 11
a. Robo1 and srGAP 11
b. The signal partners, Abl and Ena, of Slit2-Robo1 11
(4) The promoter region of Robo1 is associated with tumor 12
3. Purpose of this study 13
Material and Method 14
1. Quantitation of mRNA levels of ROBO1 using RT-PCR 15
2. Cell culture and cell lines used 15
3. Synchronization of HeLa cells and RNA preparation 15
4. Cell differentiation 16
5. Signal pathway inhibition 16
6. Construction of the expression vector 16
7. RNA interference (RNAi) 17
8. Construction of a retroviral vector delivery of Robo1B-
siRNA 18
9. Establishment Robo1 siRNA stable lines in HeLa cells 18
10. Liu’s stain 18
11. Phaloidin stain 19
12. Cell proliferation assay 19
13. Transwell assay 19
14. Three-dimensional collagen assay 20
Result 21
1. Differential mRNA expression of Robo1A and Robo1B
in fetal and adult tissues 22
2. Differential mRNA expression of Robo1A and Robo1B
in tumor and normal tissues 22
3. The mRNA expression of Robo1A and Robo1B in
cell cycle progression 23
4. The expression of Robo1A and Robo1B in cell
differentiation 23
5. The MEK1 signal inhibitor reduced Robo1B expression 23
6. Robo1B knockdown by siRNA 24
7. Robo1B siRNA induced morphological changes 24
8. Robo1B knockdown induced differentiation marker
expression 25
9. The change in proliferation rate of HeLa cells induced
by Robo1B siRNA 25
10. Effects of Robo1B siRNA in the motility and invasion
ability of HeLa cell 25
11. The identification of novol splicing variants of Robo1B
`mRNA in liver tissues 25
Discussion 27
1. Robo1A and Robo1B are temporally and spatially regulated
and differentially expressed in HCC 28
2. Robo1B overexpression may be importance in cancer
cell proliferation 28
3. Knockdown the Robo1B level leads HeLa cell
Differentiation 29
4. Knockdown of Robo1B may lead to reorganization of
cytoskeleton and decrease the malignant characters of
HeLa cell 29
5. The dissection of the Robo1B pathway: potential role of
the MAPK signal pathway 31
Reference 33
Table and Figures 40
Appendix 62
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