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研究生:唐品捷
研究生(外文):Tang, Pin-Chieh
論文名稱:GRAMD1B在肺癌中癌幹性和癌轉移所扮演之角色
論文名稱(外文):The Role of GRAMD1B in Lung Cancer Stemness and Metastasis
指導教授:洪澤民
指導教授(外文):Hong, Tse-Ming
口試委員:陳玉玲林建中
口試委員(外文):Chen, Yuh-LingLin, Chien-Chung
口試日期:2023-07-24
學位類別:碩士
校院名稱:國立成功大學
系所名稱:臨床醫學研究所
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:2023
畢業學年度:111
語文別:英文
論文頁數:59
中文關鍵詞:肺癌GRAMD1B腫瘤幹細胞癌轉移
外文關鍵詞:Lung cancerGRAMD1Bstemnessmetastasis
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肺癌是全世界死亡率最高的癌症,因為患者在確診時已處於晚期。高轉移率使其成為最致命的人類癌。因此,了解癌症進展和轉移的分子機制對於改善治療和預後至關重要。膽固醇是細胞膜的重要組成部分,對於多種細胞功能都是必需的。一些研究顯示,高水平的膽固醇與不同類型的癌症的不良預後有關。研究表明GRAMD1B是一種脂質結合蛋白,在癌細胞中已被證實是膽固醇轉運和代謝的調節因子。然而,GRAMD1B在非小細胞肺癌中的角色尚不清楚。在我們的實驗中,我們確定GRAMD1B與肺腫瘤和不良的臨床預後呈正相關。此外,與低轉移的CL1-0細胞相比,GRAMD1B在高轉移的CL1-5肺癌細胞中高度表達。此外,通過功能性實驗,我們發現GRAMD1B在非小細胞肺癌細胞中促進了細胞遷移、細胞的非貼附性生長和癌幹性特性。最後,我們試圖探索由GRAMD1B調節的分子機制,並通過RNAseq的通路富集分析和GRAMD1B過度表達的CL1-0細胞中膽固醇酯的增加,我們發現GRAMD1B可能調節膽固醇平衡。通過ACAT1抑制劑avasimibe抑制膽固醇酯化,可以抑制CL1-5和GRAMD1B過度表達的CL1-0細胞的細胞遷移。結論是這項研究確立了在非小細胞肺癌中,GRAMD1B透過ACAT1和NPC1L1調控膽固醇平衡,促進了肺癌細胞的遷移、非貼附性生長和癌幹性特性。
Globally, lung cancer is the leading cause of cancer death because of the high rate of metastasis. Thus, understanding the molecular mechanisms underlying cancer progression and metastasis is important to improve the treatment and prognosis. Cholesterol is an essential component of cell membranes and is required for various cellular functions. Several studies have shown that high levels of cholesterol are associated with poor prognosis in various types of cancer. GRAMD1B, a lipid-binding protein, has been identified as a regulator of cholesterol trafficking and metabolism in cancer cells. However, the role of GRAMD1B in non-small cell lung cancer is unknown. In this study, we identified that GRAMD1B was positively correlated with lung tumors and poor clinical outcomes. Moreover, GRAMD1B was highly expressed in high metastatic CL1-5 lung cancer cells, compared to low metastatic CL1-0 cells. Furthermore, by functional assays, we found that GRAMD1B promoted cell migration, anchorage-independent cell growth, and cancer stemness property in NSCLC cells. Finally, we tried to explore the molecular mechanisms regulated by GRAMD1B and found that GRAMD1B may regulate cholesterol homeostasis according to the pathway enrichment analysis of RNAseq results and the increase of cholesterol ester in GRAMD1B-overexpressed CL1-0 cells. Inhibition of the esterification of cholesterol by an ACAT1 inhibitor, avasimibe, suppressed cell migration of CL1-5 and GRAMD1B-overexpressed CL1-0 cells. In conclusion, this study established that GRAMD1B promoted lung cancer cell migration, anchorage-independent growth, and stemness property by regulating cholesterol homeostasis through ACAT1 and NPC1L1 in NSCLC.
中文摘要 I
Abstract II
Acknowledgement III
Contents V
Abbreviations X
Introduction 1
Lung cancer 1
Cancer metastasis 1
Cancer stem cells (CSCs) 2
Cholesterol 3
GRAMD1B 4
Rationale and specific aims 6
Materials and methods 7
Cell culture 7
In vitro wound-healing assay 7
Cell proliferation 7
Sphere formation assay 8
Soft-agar colony formation 8
Cholesterol/Cholesterol Ester-GloTM Assay 8
Plasma construction 9
Transfection 9
Lentivirus infection 9
Cellular RNA extraction 10
Quantitative real-time polymerase chain reaction (RT-qPCR) 11
Immunofluorescence by confocal microscopy 11
Western blot analysis 11
Antibodies 12
Statistical analysis 12
Results 13
GRAMD1B expression in lung tumors is correlated with poor clinical outcomes. 13
Overexpression of GRAMD1B promotes cancer cell migration. 14
Knockdown of GRAMD1B inhibits cancer cell migration. 14
GRAMD1B regulates anchorage-independent cell growth in NSCLC cells. 15
GRAMD1B induces cancer stemness in NSCLC. 15
GRAMD1B is correlated to cholesterol homeostasis, lung cancer survival, stemness and metastasis signatures in NSCLC cells. 16
GRAMD1B locates on plasma membrane and endoplasmic reticulum in NSCLC cells and cholesterol induces the accumulation of GRAMD1B, forming the spots in cells. 16
Cholesterol and its metabolite, 27-hydroxycholesterol, affect the cell migration ability in NSCLC cells. 17
Overexpression of GRAMD1B upregulated total cholesterol amount in CL1-0 cells through the increase of cholesterol ester. 17
Avasimibe reduces the cell migration ability in CL1-5 cells and GRAMD1B-overexpressed CL1-0 cells. 18
Ezetimibe reduces the free cholesterol level and the cell migration ability in GRAMD1B-overexpressed CL1-0 and CL1-5 cells. 19
Discussion 20
Conclusions 24
References 25
Figures 30
Figure 1. GRAMD1B expression is positively correlated with malignant pathways in lung tumors. 30
Figure 2. GRAMD1B expression is positively correlated with lung tumors and poor clinical outcome. 31
Figure 3. GRAMD1B is upregulated in highly invasive CL1-5 lung cancer cell line at both of the mRNA and protein levels. 32
Figure 4. Manipulating GRAMD1B expression in CL1-0 and CL1-5 cells. 33
Figure 5. Overexpression of GRAMD1B has no significant effect on NSCLC cell growth. 34
Figure 6. GRAMD1B promotes lung cancer cell migration in vitro. 35
Figure 7. Knockdown of GRAMD1B has no significant effect on NSCLC cell growth. 36
Figure 8. GRAMD1B knockdown decreases lung cancer cell migration in vitro. 37
Figure 9. GRAMD1B overexpression significantly induces anchorage-independent cell growth in CL1-0 cells. 38
Figure 10. GRAMD1B knockdown significantly reduces anchorage-independent cell growth in CL1-5 cells. 39
Figure 11. GRAMD1B overexpression promotes cancer stemness property in CL1-0 cells. 40
Figure 12. GRAMD1B knockdown reduces cancer stemness property in CL1-5 cells. 41
Figure 13. The top 20 enriched pathways by RNA sequencing analysis of GRAMD1B-knockdown CL1-5 cells. 42
Figure 14. RNA sequencing data analysis of GRAMD1B-knockdown CL1-5 cells shows the correlation between GRAMD1B and cholesterol homeostasis, lung cancer survival, stemness, and metastasis. 43
Figure 15. GRAMD1B-knockdown cells show an epithelial phenotype. 44
Figure 16. GRAMD1B locates on plasma membrane and ER. 45
Figure 17. Cholesterol treatment increases the number of GRAMD1B fluorescence spots in GRAMD1B- overexpressed CL1-0 cells. 46
Figure 18. Cholesterol promotes the CL1-0 cell migration. 47
Figure 19. Cholesterol promotes the CL1-5 cell migration. 48
Figure 20. 27-Hydroxycholesterol does not affect the CL1-0 cell motility. 49
Figure 21. 27-Hydroxycholesterol promotes the CL1-5 cell motility. 50
Figure 22. The levels of total cholesterol, free cholesterol and cholesterol ester in CL1-0 cells with GRAMD1B overexpression or combined with avasimibe treatment. 51
Figure 23. Effect of avasimibe on cholesterol levels in CL1-5 cells. 53
Figure 25. Avasimibe inhibits the CL1-5 cell motility. 55
Figure 26. Ezetimibe decreases the levels of total and free cholesterol in GRAMD1B-overexpressed CL1-0 cells. 56
Figure 27. Ezetimibe decreases the levels of total and free cholesterol in CL1-5 cells. 57
Figure 29. Ezetimibe inhibits the CL1-5 cell motility. 59
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