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研究生：	李蓉蓉 Li, Jung-Jung
論文名稱：	針對SCUBE3篩選適體的影響及應用 The effect and application of aptamer against SCUBE3
指導教授：	洪澤民 Hong, Tse-Ming
學位類別：	碩士 Master
系所名稱：	醫學院 - 臨床醫學研究所 Institute of Clinical Medicine
論文出版年：	2016
畢業學年度：	104
語文別：	英文
論文頁數：	62
中文關鍵詞：	SCUBE3 、適體、肺癌
外文關鍵詞：	SCUBE3, Aptamer, lung cancer
相關次數：	點閱：145 下載：1
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Signal peptide-CUB-EGF-like domain-containing protein 3 (SCUBE3)是一個經過糖修飾後的分泌型蛋白，而SCUBE3在肺癌檢體中有大量的
表現，並與上皮-間質轉換(EMT)和轉移有關。因此我們希望可以發現與SCUBE3結合的抑制劑，藉此作為有效的標靶治療。適體(Aptamers)為一單股DNA或RNA，且被發現可以與其目標分子具有專一性的結合，而近年來也被利用在診斷及治療方面。因此我們希望可以發展出可以與SCUBE3專一性結合的適體，並利用適體來抑制SCUBE3在肺癌中所誘導的腫瘤形成能力，近而發展出新的肺癌治療方法。
首先我們利用硝化纖維素膜，來進行指數富集的配體有系統演化(SELEX)的實驗，藉此篩選出可以與SCUBE3結合的適體。之後，我們將這些適體定序，並找出最主要且占多數的適體，並命名為S3-A2、AP-NS3-1、AP-NS3-2、AP-NS3-3及AP-NS3-4。然後將這些適體利用免疫螢光染色的實驗來偵測適體與SCUBE3的專一性結合力，我們發現這些適體比控制組較容易與大量表現SCUBE3的細胞結合。此外我們利用傷口癒合爬行實驗來觀察適體對人類臍帶靜脈內皮細胞和非小型細胞肺癌細胞爬行能力的影響；且也利用μ-Slide Angiogenesis來探討在人類臍帶靜脈內皮細胞中，適體處理後血管生成的能力。我們發現其中的AP-NS3-1及AP-NS3-3可以抑制人類臍帶靜脈內皮細胞的爬行能力和管狀生成能力，甚至可以減少非小型細胞肺癌細胞的爬行數目。緊接著我們也發現AP-NS3-1及AP-NS3-3可能是透過調節第一型類胰島素生長因子受體(IGF1R)，進而影響下游的訊號，像是Akt及Erk，最終導致癌轉移能力被抑制。最後我們利用了低貼覆性的六孔盤，來觀察適體處理後，非小型細胞肺癌細胞的球體形成能力。由結果我們也發現了S3-A2、AP-NS3-2及AP-NS3-4不但可以抑制Hop62細胞球體生成的大小，也可以減少生成數目。根據上述的結果，發展出與SCUBE3專一性結合的適體可以抑制腫瘤的血管生成和癌症的轉移，並成為在肺癌治療中的有潛力的應用。

Signal peptide-CUB-EGF-like domain-containing protein 3 (SCUBE3) is a secreted cell-surface glycoprotein. SCUBE3 is overexpressed and is correlated with epithelial-mesenchymal transition (EMT) and cancer metastasis of lung cancer cells. These findings suggest that SCUBE3 might be a potential oncotarget for pharmacological intervention. Therefore, we wanted to find some SCUBE3-specific inhibitors. Aptamers are single strand DNA or RNA, which are selected to tightly and specifically bind to target molecules. In recent years, aptamers can be coupled to diagnostic or therapeutic agents. Accordingly, we developed the SCUBE3 specific aptamers, which can effectively suppress the SCUBE3-mediated function in lung cancer progression, for future applications, especially for lung cancer therapy. First, we performed nitrocellulose membrane filtration-based systematic evolution of ligands by exponential enrichment (SELEX) to select aptamers against SCUBE3. After several rounds of SELEX, the selected aptamers were amplified by PCR and named as S3-A2, AP-NS3-1, AP-NS3-2, AP-NS3-3, and AP-NS3-4. Then we verified the specific binding between aptamers and SCUBE3-expressed cells by immunofluorescence. We found that those aptamers preferentially bound to SCUBE3-expressed cells, compared to non-SCUBE3-expressed cells. Moreover, we analyzed the effect of the SCUBE3 aptamers in immortalized HUVECs or NSCLC cells by cell viability assay, wound healing migration assay and μ-Slide Angiogenesis assay. We found that while the SCUBE3 aptamers did not affect the cell viability, AP-NS3-1 and AP-NS3-3 inhibited the ability of migration and the tube formation in immortalized HUVECs and repressed the cell migration of CL1-5. To investigate the possible mechanism of AP-NS3-1 and AP-NS3-3 in suppressing cell motility, we found that AP-NS3-1 and AP-NS3-3 suppressed the IGF1R activation and then decreased the downstream signaling, Erk and Akt. Finally, we observed the ability of sphere formation in SCUBE3 aptamer-treated NSCLC cells in low-attachment 6-well plates. The result showed that S3-A2, AP-NS3-2 and AP-NS3-4 decreased the number of sphere in Hop62 cells. According to above results, we suggested that AP-NS3-1 and AP-NS3-3 targeting SCUBE3 repress tumor angiogenesis and the motility of NSCLC cells in vitro, potentially as cancer therapeutic drugs. Moreover, S3-A2, AP-NS3-2 and AP-NS3-4 may be useful aptamers in suppressing cancer stemness. Taken together, SCUBE3-specific aptamers may suppress tumor angiogenesis, cancer migration/metastasis and cancer stemness and be potential tools for lung cancer therapy.

Contents
摘要	I
Abstract	III
Acknowledgement	V
Contents	VII
Abbreviation	X
Introduction	1
Lung cancer	1
Aptamers	2
Therapeutic applications of aptamers	3
SCUBE3	4
SCUBE3 in cancers	5
Tumor metastasis	5
Rationale and Specific aims	8
Material and Methods	9
1.	Immunohistochemistry (IHC)	9
2.	Nitrocellulose membrane filtration-based SELEX	9
3.	Polymerase chain reaction (PCR)	10
4.	Agarose gel electrophoresis	10
5.	ssDNA isolation	10
6.	Immunofluorescence	11
7.	Cell viability- WST-1 assay	12
8.	Wound healing assay	12
9.	Tube formation	12
10.	Spheroid cultivation	13
11.	Western Blot	13
12.	RNA purification	13
13.	Reverse transcription (RT)-PCR	14
14.	Quantitative real-time PCR (qPCR)	14
15.	Cell-based affinity assay	14
Results	16
1.	The expression of SCUBE3 is correlated with tumor stage and metastasis in lung cancer.	16
2.	DNA aptamers are selected against SCUBE3 with high specificity.	16
3.	The selected aptamers can bind to endogenous SCUBE3.	18
4.	The SCUBE3-specific aptamers bind to Hop62 cells with Kd values in a nanomolar range.	19
5.	SCUBE3-specific aptamers, S3-A2, AP-NS3-1 and AP-NS3-3 inhibit cell migration and tube formation in immortalized human umbilical vein endothelial cells (I-HUVECs).	20
6.	SCUBE3-specific aptamers, AP-NS3-1 and AP-NS3-3, significantly repress the migration ability of CL1-5 cells.	21
7.	SCUBE3-specific aptamers, S3-A2, AP-NS3-2 and AP-NS3-4, suppress the ability of sphere formation in Hop62 cells.	22
8.	AP-NS3-1 and AP-NS3-3 may repress the migration ability through blocking the IGF1R signaling.	23
9.	S3-A2, AP-NS3-2 and AP-NS3-4 may suppress the ability of sphere formation through decreasing the ALDH1A1 expression.	23
Discussion	25
Conclusion	28
References	29
Figures	34
Figure 1. SCUBE3 is correlated with tumor stages and metastasis in lung cancer.	34
Figure 2. The procedure and result of SELEX	35
Figure 3. The SCUBE3-specific aptamers are selected by membrane SELEX and bind to SCUBE3 with high specificity.	38
Figure 4. The SCUBE3-specific aptamers bind to SCUBE3 with high specificity and affinity by immunofluorescence.	43
Figure 5. The SCUBE3-specific aptamers bind to SCUBE3 with Kd values in the nanomolar range by ELISA.	45
Figure 6. The SCUBE3-specific aptamers suppress the abilities of cell migration and tube formation, but not cell viability, in endothelial cells.	48
Figure 7. The SCUBE3-specific aptamers can inhibit the ability of cell migration but not affect the cell viability in CL1-5 cells.	50
Figure 8. The SCUBE3-specific aptamers can suppress the ability of sphere formation but not cell viability in Hop62 cells.	52
Figure 9. AP-NS3-1 and AP-NS3-3 reduce the IGF1R signaling.	54
Figure 10. S3-A2, AP-NS3-2 and AP-NS3-4 decrease the ALDH1A1 mRNA level.	55
Figure 11. Summary diagram of the effect of SCUBE3-specific aptamers in cells.	56
Tables	57
Table 1. Deparaffination	57
Table 2. Retrieval buffer	57
Table 3. SELEX buffer	57
Table 4. SELEX condition in each round	58
Table 5. Condition in PCR	58
Table 6. SSC buffer	58
Table 7. Running buffer and Transfer buffer	59
Table 8. TBS buffer and TBS-T buffer	59
Table 9. RT-PCR	59
Table 10. qPCR	60
Table 11. The relation between the expression of SCUBE3 and clinical features in lung cancer patients	61
Table 12. SCUBE3 aptamer NGS output	62


                                    

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校內：2021-08-23公開
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