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研究生：	陳佳微 Chen, Chia-Wei
論文名稱：	在非小細胞肺癌中與轉移相關的長鏈非編碼核糖核酸之功能性分析 Functional analysis of metastasis-associated long non-coding RNAs in non-small cell lung cancer
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：	2021
畢業學年度：	109
語文別：	英文
論文頁數：	63
中文關鍵詞：	肺癌、長鏈非編碼核糖核酸、癌轉移、腫瘤幹細胞
外文關鍵詞：	lung cancer, long noncoding RNAs (lncRNAs), cancer metastasis, cancer stem cells
相關次數：	點閱：219 下載：0
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肺癌由於缺乏早期診斷與在晚期會發生轉移，長久以來是全世界最常見也是致死率最高的癌症，長鏈非編碼核糖核酸一般定義為轉錄長度大於兩百個核苷酸並不具有轉譯成蛋白質的特性，而越來越多的證據已經指出長鏈非編碼核糖核酸在癌症形成中扮演一個很重要的調控腳色並且對於疾病的診斷與癌症的治療是很有潛力的標的物。然而，促進癌症轉移的詳細機制仍尚未被清楚的研究。在我們的實驗中，我們找出一個與轉移有關聯且在肺癌中表現量與較低存活率有相關性地的長鏈非編碼核糖核酸U21，透過抑制與放大U21的表現量做功能分析，我們發現U21在腫瘤進展過程中扮演治癌的腳色，抑制U21會降低較惡化的非小细胞肺癌細胞的爬行與侵襲的能力。除了會促進癌轉移的功能，我們也發現U21會參與在腫瘤幹細胞的特性中。最後我們也剖析U21調控的分子機制並找到與之相互作用的目標，包含了微小核醣核酸與蛋白質，長鏈非編碼核糖核酸U21與相互作用的蛋白質所參與的治癌性機制與下游相關的目標都將被找出。結論是在我們的研究證實了一個新穎並具有致癌特性的長鏈非編碼核糖核酸會參與在肺癌的進展當中。

Owing to the lack of early diagnosis and metastasis at late stage, lung cancer is the most common cancer and the leading cause of cancer death worldwide. Long noncoding RNAs (lncRNAs) are generally defined as transcripts longer than 200 nucleotides without protein coding potential, and accumulating evidence have indicated that lncRNAs are important regulators of tumorigenesis and as potential biomarkers for the disease diagnosis and cancer treatment. However, the underlying mechanisms that promote cancer metastasis remain unclear. Here, we identified a metastasis-related lncRNA U21, which expression was correlated with poor survival in lung cancer patients. According to the results from the loss- and gain-of-function assays, we found that lncRNA U21 had an oncogenic role in tumor progression. LncRNA U21 knockdown inhibited the abilities of cell migration, invasion, and proliferation in malignant non-small cell lung cancer (NSCLC) cells. In addition to the metastasis-promoting role, lncRNA U21 was involved in cancer stemness as well. Finally, we dissected the molecular mechanisms regulated by lncRNA U21 and found the molecular interaction between lncRNA U21 and its potential targets including miRNAs and proteins. The lncRNA U21-interacted proteins and the tumorigenic mechanisms of lncRNA U21 and its associated targets will be further elucidated. This study revealed that lncRNA U21 exhibits a novel oncogenic role in regulating the progression of lung cancer.

中文摘要	I
Abstract	II
致謝	III
Table of Contents	V
Abbreviations	IX
Introduction	1
Lung cancer	1
Cancer metastasis	1
Cancer stem cells (CSCs)	3
Long non-coding RNAs (lncRNAs)	4
LncRNAs associate with cancer metastasis and CSCs	5
Rationale and specific aims	8
Materials and Methods	9
Cell culture	9
In vitro wound healing assay	9
Cellular RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)	9
Western blot analysis	10
Lentivirus infection	11
Cell proliferation	11
Plasmid construction	12
Sphere assay	12
Limited dilution	12
ALDEFLUOR™ assay	13
Soft-agar colony formation	13
Separation of RNA nuclear and cytoplasmic fractions	14
RNA gel electrophoresis	14
RNA pull-down assay for interacted miRNA detection	15
RNA pull-down assay for interacted protein detection	16
Silver staining	16
Transwell invasion assay	17
Cell tracking	17
Statistical analysis	18
Results	19
LncRNA U21 expression is abundant in highly metastatic cells and correlated with poor survival in lung cancer.	19
Overexpression of lncRNA U21 has no effect on cancer cell proliferation and migration.	20
Knockdown of lncRNA U21 inhibits cancer cell proliferation, migration and invasion.	20
The expression of EMT markers in lncRNA U21-manucipated NSCLC cells.	21
Anchorage-independent growth in lncRNA U21-overexpressed NSCLC cells.	21
LncRNA U21 induces cancer stemness.	22
The expression of CSC markers in lncRNA U21-manucipated NSCLC cells.	22
Identification of the lncRNA U21-interacted miRNAs through RNA pull-down.	23
Identification of the lncRNA U21-interacted proteins through RNA pull-down.	24
Discussion	26
Conclusions	31
References	32
Tables	37
Figures	38
Figure 1. CL1-5 cells with highly migratory and invasive ability are selected from CL1-0 cells.	38
Figure 2. LncRNA U21 is markedly upregulated in metastatic cells and associated with poor survival in lung cancer.	39
Figure 3. Establishment of lncRNA U21 over-expression in CL1-0 cells and lentiviral-based lncRNA U21 knockdown system in CL1-5 cells.	40
Figure 4. Overexpressed lncRNA U21 has no significant effect on NSCLC cell growth.	41
Figure 5. Overexpressed lncRNA U21 has no significant effect on migration of NSCLC cells.	42
Figure 6. Knockdown of lncRNA U21 inhibits NSCLC cell growth.	43
Figure 7. Knockdown of lncRNA U21 inhibits NSCLC cell migratory and invasive capability.	44
Figure 8. Knockdown of lncRNA U21 inhibits NSCLC cell motility.	45
Figure 9. The expression of EMT markers in lncRNA U21-overexpressed CL1-0 and lncRNA U21-knockdown CL1-5 cells.	46
Figure 10. LncRNA U21 overexpression promotes the anchorage-independent growth of cancer cells.	47
Figure 11. LncRNA U21 is upregulated under the sphere formation condition in CL1-0 cells.	48
Figure 12. Overexpressed lncRNA U21 augments CSC phenotype.	49
Figure 13. Knockdown of lncRNA U21 attenuates CSC phenotype.	50
Figure 14. The expression of stemness markers in lncRNA U21-overexpressed CL1-0 and lncRNA U21-knockdown CL1-5 cells.	51
Figure 15. ALDH activity in lncRNA U21-knockdown NSCLC cells.	53
Figure 16. The expression of stemness-related transcription factors in lncRNA U21-knockdown NSCLC cells under sphere formation condition.	54
Figure 17. LncRNA U21 is predominantly localized in the cytoplasm.	55
Figure 18. Detection of RNA and protein components of the MS2 complexes in GST pulldown.	56
Figure 19. LncRNA U21-associated miRNAs.	58
Figure 20. The associated protein candidates are identified via lncRNA U21 RNA pull-down.	59
Appendix	60
Appendix 1. pcDNA 3.1 (+) plasmid map for lncRNA U21 overexpression.	60
Appendix 2. pLKO TRC001 plasmid map for lncRNA U21 knockdown.	61
Appendix 3. pcDNA3-MS2-contained plasmid map for identification of lncRNA U21-inteacted miRNAs.	62
Appendix 4. pCI-neo plasmid map for detection of lncRNA U21-associated proteins.	63
                                    

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2026-07-29公開

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