臺灣博碩士論文加值系統

細胞移動的初步機制，必須在基材上進行延展。細胞延展在癌細胞轉移中扮演着一個關鍵性的調控步驟。因此透過調控細胞延展的研究可以提供防止細胞轉移的治療策略。細胞延展和爬行都涉及actin聚合在細胞膜的前緣和尾部抽離。調節細胞延展和爬行雖已被廣泛研究，但詳細分子機制仍不清楚。根據以往的研究，Mob2蛋白會藉由參與actin細胞骨架的重新排列進而影響神經纖維生長。此外，Mob2在酵母菌的極性生成和果蠅感光細胞的發育過程中也影響actin細胞骨架。但是目前沒有研究指出Mob2是否會影響細胞延展和細胞爬行功能。在本研究中，我們發現人類纖維腫瘤(HT1080) 細胞展延需要hMOB2蛋白參與。免疫細胞染色結果顯示，人類纖維腫瘤(HT1080) 細胞爬行時，hMOB2會分佈在爬行的細胞前緣。進一步探討hMOB2是否參與細胞移動，我們利用shRNA的方式降低細胞內hMob2蛋白的表現觀察細胞延展和細胞爬行的情況，hMOB2在人類纖維腫瘤(HT1080) 細胞會延遲細胞展延。除此之外，過量表達野生型hMOB2蛋白會促進hMOB2累積在細胞的前緣和增強細胞延展能力。為了更進一步瞭解hMOB2在細胞移動時的功能性區域，我們建立了持續表達突變型（A107G、Y110A）的hMOB2蛋白。結果顯示突變型的hMOB2蛋白會影響hMOB2蛋白在細胞前緣的累積及延遲細胞延展的能力。綜合以上結果，推測hMOB2蛋白會透過影響其表现在細胞前緣，來調控細胞展延。然而在人類纖維腫瘤細胞 (HT1080)移動的速率，降低細胞內hMob2，表現野生型和突變型的細胞株中，細胞移動的速率並沒有顯著的差異。過量表達野生型hMOB2蛋白會形成較多的板狀偽足結構，並且會以群體方式來移動。此研究結果提供對細胞延展的分子機制有更進一步的了解。

Cell spreading is an initial mechanism for cell migration which plays a vital role in cancer development. Cell spreading has been shown to act as one of the key regulating steps between static and metastatic transition of a cancer cell. Hence, by identifying regulatory networks controlling cell spreading, it may provide valuable information and therapeutic strategies for preventing tumor metastasis. Both cell spreading and cell migration involve actin polymerization at the leading edge of plasma membrane follow by cell retraction at the rear end of cells. The molecular mechanisms in regulating cell spreading and cell migration have been extensively studied but remain unclear. Studies from yeast, Drosophila to mammalian cells have shown that MOB2 protein plays an important role in controlling the cell morphology changes by affecting cell polarity and rearrangement of actin cytoskeleton. Currently there is no research done to study the function of Mob2 in cell spreading and cell migration. In this study, we identified hMOB2 protein which plays a significant role in promoting cell spreading in HT1080 human fibrosarcoma cells. Our results showed that hMOB2 was detected at the leading edge of migrating HT1080 human fibrosarcoma cell. To study whether hMOB2 was involved in cell motility, we downregulated hMOB2 expression using RNA interference and found that cell spreading was delayed in HT1080 cells. In addition, we observed that overexpression of hMOB2 enhanced cell spreading in HT1080 cells and enhanced its accumulation at the leading edge. Furthermore, to determine the possible functional domain in cell motility, we successfully generated A107G, Y110A point mutated hMOB2 stable cell lines. Over-expressed point mutated hMOB2 expression delayed cell spreading and suppressed its accumulation at the leading edge. These observations suggested that hMOB2 affects cell spreading by regulating its expression at leading edge. No significant difference was observed in the migration rate between the different HT1080 cell populations when the percentage of gap closure was determined. However, over-expressed wild type hMOB2 induced broad lamellipodial structures and moved as a coherent group when compared with parent cells. These studies provided additional information on the molecular mechanisms which control cell spreading.

TABLE OF CONTENTS

Page

ABSTRACT 1
摘要 3
CHAPTER
1.0 INTRODUCTION
1.1 Cell spreading 5
1.2 Cell Migration 6
1.3 Signal involves in actin polymerization 6
1.4 MOB protein 8
1.4.1 MOB structure 9
1.4.2 Mob2 in yeast study 10
1.4.3 Dmob2 in Drosophila study 10
1.4.4 Mob2 in mammalian cells study 11
1.5 Specific aims on my studies 12

2.0 MATERIALS AND METHODS
2.1 Culture HT1080 cells 14
2.2 Mob2 RNA interference 14
2.3 Infection of HT1080 cells 14
2.4 Construction of plasmids
2.4.1 RNA extraction 15
2.4.2 Reverse transcription-Polymerase Chain Reaction 16
2.4.3 Restriction endonucleases 17
2.4.4 Ligation 17
2.4.5 Bacterial Transformation 17
2.4.6 Plasmid DNA extraction 18
2.5 Transfection of HT1080 cells 18
2.6 Western Blot 19
2.7 Immunofluorescence 20
2.8 Wound healing assay 21
2.9 Cell spreading 21
2.10 Cell proliferation assay and population doubling time 22

3.0 RESULTS
3.1 hMOB2 expression in HT1080 cells 23
3.2 Downregulation of hMOB2 affects cell spreading 23
3.3 Overexpression of hMOB2 promotes its accumulation at the 25
leading edge of migrating cells and enhances cell spreading
3.4 Structural conserved of Mob2 proteins 27
3.5 A107G,Y110A point mutate hMOB2 interferes cell spreading 28
3.6 Qualitative changes in overexpression hMOB2 migrating cells 30


4.0 DISCUSSION
4.1 Cell spreading Classification 33
4.2 hMOB2 functions at the plasma membrane 34
4.3 Point mutation hMOB2 affects localization of hMOB2 36
4.4 hMOB2 does not affect cell migration but affected migrating 37
morphology
4.5 Partner interacting with hMOB2 38

5.0 REFERENCES 40

LIST OF FIGURES
Fig. 1. Sequence alignment of human and mouse Mob2 proteins. 45
Fig. 2. Subcellular localization of hMOB2 in HT1080 cells. 46
Fig. 3. Downregulation of hMOB2 expression in 47
shRNA treated HT1080 cells.
Fig. 4. Downregulation hMob2 does not affect the localization 48
of actin cytoskeleton in HT1080 cells.
Fig.5. Three categories of spreading cells. 49
Fig. 6. Downregulation of hMOB2 expression affected cell spreading. 50
Fig. 7. Protein sequence alignment of generated wild type hMOB2. 54
Fig. 8. Overexpression of wild type human MOB2 stable cell lines. 55
Fig. 9. Overexpression of wild type hMOB2 promoted 56
its accumulation at the leading edge of migrating cells.
Fig. 10. Overexpression of hMOB2 spreads faster than 57
wild type (parent cells).
Fig. 11. Sequence alignment of human MOB proteins. 60
Fig. 12. Experimental design on point mutation and deletion region 61
of hMOB2 construct.
Fig. 13. Protein sequence alignment of A107G,Y110A point mutation 62
hMOB2 construct.
Fig. 14. Overexpression of A107G,Y110A point mutation hMOB2 63
stable cell lines.
Fig. 15. Expression of A107G,Y110A point mutation hMOB2 64
protein suppressed its accumulation at the leading edge.
Fig. 16. Point-mutated hMOB2 protein interfered cell spreading. 65
Fig. 17. Actin filament enriched at the plasma membrane of HT1080 cells 67
expressed hMOB2.
Fig. 18. Wound healing assay shown similar migration rate. 68
Fig. 19. Qualitative changed in overexpression hMOB2 expressing cells 70
in cell migration.
Fig. 20. Higher magnification indicated qualitative changes in 71
overexpression hMOB2 cells in wound healing assay.
Fig. 21. Cell proliferation assay. 72

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