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研究生:吳孟哲
研究生(外文):Meng-Che Wu
論文名稱:探討mCostars在肺癌細胞中表現之調控
論文名稱(外文):Exploring the Regulation of mCostars Expression in Lung Cancer Cells
指導教授:陳美瑜陳美瑜引用關係
指導教授(外文):Mei-Yu Chen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:英文
論文頁數:37
中文關鍵詞:肺癌
外文關鍵詞:mCostarsMicroRNAlung cancer
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mCostars (mammalian Costars)為Costars於哺乳類動物中的同源蛋白;Costars是我們實驗室首先從一個趨化運動異常黏菌突變株中發現的。過去實驗室的研究成果發現將mCostars送入缺乏Costars的黏菌細胞株內,可以拯救細胞移動及肌動蛋白的缺陷現象,因此我們認為mCostars與Costars在功能上是具有高度保留性的。我們也發現在臨床的肺癌檢體上,mCostars是被大量表現的,而將mCostars 做knock down時發現細胞移行能力會下降。這些實驗結果說明mCostars可能在細胞移行及癌化方面扮演重要角色,但詳細機制目前仍不明瞭。在本篇研究中,我們嘗試探討mCostars的表現調控機制,而著重於由microRNA控管的轉錄後修飾階段。MicroRNAs為內源性且不具備轉譯功能的小片段RNAs,已知miRNAs會參與許多細胞生化反應過程。透過生物資訊搜尋的方式,我們鑑定到一組候選miRNAs,其種子序列位於mCostars的3'UTR中。我們在肺癌細胞株和臨床標本中對感興趣的miRNAs進行表現分析,以評估mCostars和這些miRNA的表現之間有無關聯。也透過於肺癌細胞中過度表現或抑制這些miRNAs來測試這些miRNAs對mCostars表現的調控作用。並以螢光素酶報導基因測定研究這些miRNAs是否可直接靶向mCostars的3'UTR。我們的結果證實了mCostars和miRNAs之間的功能關聯性,並且提供了mCostars參與的細胞移行可能受控於miRNAs調控的假設依據。
mCostars (mammalian Costars) is a mammalian homologue of Costars; Costars was first discovered in our laboratory from a chemotaxis-defective Dictyostelium mutant. Previous results in our laboratory have demonstrated that mCostars can rescue the motility and actin defects in Dictyostelium cells lack of Costars, indicating that mCostars and Costars are functionally conserved. We have also found that mCostars is highly expressed in clinical lung cancer tissues compared to the normal parts, and mCostars-silenced cells show decreased migration activity. These findings suggest that mCostars plays a crucial role in cell migration and even in tumorigenesis; however, the underlying mechanism is still not clear. In this study, we investigated the expression control of mCostars, focusing on microRNA-mediated post-transcriptional regulation. MicroRNAs are endogenous, small non-coding RNAs regulating gene expression at the post-transcriptional level and are involved in many cellular processes. By bioinformatics searches we identified a group of candidate miRNAs with their seed sequences in the 3’UTR of mCostars. We performed expression profiling of the miRNAs of our interest in lung cancer cell lines and clinical specimens to assess the association of the expression of mCostars and these miRNAs. The regulatory role of these miRNAs on mCostars expression was tested by overexpressing these miRNAs or administering inhibitors of these miRNAs to lung cancer cells. Luciferase reporter assays were carried out to investigate if these miRNAs may directly target the 3’UTR of mCostars. Our results have demonstrated a functional link between mCostars and miRNAs, and suggest a regulatory role of miRNAs in mCostars-regulated cell migration.
摘要 i
Abstract iii
Contents v
List of Figures vii
Introduction 1
Costars and mCostars 1
Costars, mCostars and cell migration 1
mCostars and lung cancer 2
Functional roles of miR-145 4
Materials and Methods 6
Cell lines and cell culture 6
Prediction of mCostars-targeting miRNAs 6
Plasmid construction 6
Transfection and miRNA inhibitor treatment 7
Reporter assay 8
Protein preparation and Western blot analysis 8
RNA isolation, real-time PCR (qPCR) and microRMA detection 9
Results 10
The mRNA level of mCostars does not match protein expression in clinical samples 10
mCostars is a potential target of microRNAs 10
MiR-145 is negatively correlated with mCostars 11
MiR-145-5p downregulates mCostars and directly targets the 3’UTR 11
Discussion 14
References 17
Figures 22
Appendix 33
Table 37


List of Figures
Figure 1. The expression of mCostars mRNA in clinical samples of lung adenocarcinoma. 22
Figure 2. mCostars is a potential target of miRNAs. 23
Figure 3. MiR-145 is negatively correlated with mCostars in clinical lung adenocarcinoma samples. 24
Figure 4. MiR-145 is negatively correlated with mCostars in lung cancer cell lines. 25
Figure 5. MiR-145 downregulates mCostars 3’UTR reporter expression. 26
Figure 6. MiR-145-5p directly targets mCostars 3’UTR. 27
Figure 7. Overexpression of miR-145 decreases mCostars mRNA level in H1299 cells. 28
Figure 8. Overexpression of miR-145 decreases mCostars protein expression in H1299 cells. 29
Figure 9. Downregulation of miR-145-5p does not increase mCostars mRNA level in H838 cells. 30
Figure 10. Downregulation of miR-145-5p and miR-145-3p does not increase mCostars protein expression in H838 cells. 31
Figure 11. Downregulation of miR-145-5p does not increase mCostars 3’UTR reporter expression. 32
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