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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 陳靜宜,丁詩同 | |
| dc.contributor.author | Han-Jen Lin | en |
| dc.contributor.author | 林函臻 | zh_TW |
| dc.date.accessioned | 2021-06-08T03:27:21Z | - |
| dc.date.copyright | 2020-01-15 | |
| dc.date.issued | 2019 | |
| dc.date.submitted | 2019-12-30 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21123 | - |
| dc.description.abstract | 在家禽胚胎發育過程，卵黃為重要營養來源。其中固醇O–醯基轉移酶1（Sterol O-acyltransferase 1, SOAT1）將游離膽固醇酯化成膽固醇酯。於家禽胚胎發育後期，卵黃囊膜中SOAT1 mRNA濃度與酶活性皆隨時間顯著增高，且卵黃囊膜的膽固醇酯濃度也極高，推論膽固醇酯化與利用卵黃脂質機制息息相關。小片段核糖核酸（microRNAs, miRNAs）被認為是影響mRNA穩定性而降低其轉錄與轉譯的原因之一。發育中的家禽胚胎肝臟與肌肉皆發現有miRNAs參與調節代謝，但miRNAs對於卵黃囊膜的影響或參與其中脂質代謝的角色卻仍未知。因此利用miRNA sequencing分析入孵後不同胚胎時期卵黃囊膜中miRNA，尋找可能藉由抑制乙型轉化生長因子受體1型（transforming growth factor beta receptor type 1, TGFBR1）去影響SOAT1表現的候選miRNA，並以real time PCR確認這些miRNAs於胚胎發育各時期之表現量。研究發現gga-miR-181a-5p表現量與發育時期SOAT1 mRNA變化模式相近、gga-miR-429-3p則與其相反。在鵪鶉卵黃囊膜內胚層上皮細胞的試驗中發現，轉染5 nM的gga-miR-181a-5p、gga-miR-133a-5p與gga-miR-429-3p有效降低SOAT1、TGFBR1 mRNA表現量。通常miRNA以seed region辨識並結合到目標基因3’UTR，作為miRNA直接影響目標的證據之一。本試驗確認gga-miR-181a-5p與gga-miR-429-3p能結合至TGFBR1 3’UTR，而在突變miRNA seed region之後，gga-miR-181a-5p與gga-miR-429-3p皆失去與3’UTR之結合能力。此二miRNA轉染會顯著的降低SOAT1與TGFBR1蛋白質表現量。此外，TGFBR1抑制劑LY364947，也能有效降低SOAT1 mRNA表現量，與miRNA轉染試驗結果相符。這些結果顯示gga-miR-181a-5p與gga-miR-429-3p可能都是藉由調控TGFβ訊息傳導路徑，去影響卵黃囊膜內胚層上皮細胞內的SOAT1基因表現與其功能。所以在家禽胚胎發育期間，藉由調控卵黃囊膜中某些miRNAs表現量，就可影響卵黃囊膜對於脂質的利用能力，此種特性可能可以利用來改進禽胚的發育和孵化率。 | zh_TW |
| dc.description.abstract | Nutrients are utilized and re-constructed by endodermal epithelial cells (EECs) of yolk sac membrane (YSM) in avian species during embryonic development. Sterol O-acyltransferase 1 (SOAT1) is the key enzyme to convert cholesterol to cholesteryl ester to facilitate the delivery to growing embryos. During embryonic development, yolk absorption is concomitant with significant changes of SOAT1 mRNA concentration and enzyme activity in YSM. Expression of microRNAs (miRNAs) occurs in the embryonic liver and muscle during avian embryogenesis. However, the expression of miRNAs in YSM during embryogenesis and the involvement of miRNAs in lipid utilization are not known. Using a miRNA sequencing technique, I found several miRNA candidates and confirmed their expression patterns individually by real time PCR. MiRNA candidates were selected based on the expression pattern and their possible roles in respect to transforming growth factor beta receptor type 1 (TGFBR1) that regulates the function of SOAT1. Similar to SOAT1 mRNA, gga-miR-181a-5p expression was gradually elevated during embryonic development, whereas of gga-miR-429-3p expression in YSM was gradually decreased during embryonic development. Inhibitory effects of gga-miR-181a-5p or gga-miR-429-3p on the potential targets (SOAT1 and TGFBR1) at both in transcript and protein level were further observed by transient miRNA transfections in EECs. Mutated TGFBR1 3’UTR failed to direct pairings of gga-miR-181a-5p and gga-miR-429-3p. Treatment of TGFBR1 inhibitor, LY364947, further decreased SOAT1 transcription. Similar results were also observed by the miRNA transfection studies. The results showed a vital role of gga-miR-181a-5p and gga-miR-429-3p in regulating TGFβ pathway, further affecting downstream SOAT1 expression and function in the YSM, and therefore suggesting a critical miRNA-mRNA regulation of avian yolk lipid utilization in YSM. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T03:27:21Z (GMT). No. of bitstreams: 1 ntu-108-F00626001-1.pdf: 9681186 bytes, checksum: d26742394d60211049322a2b45c4b146 (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | 目次Contents
國立臺灣大學博士學位論文口試委員會審定書 i 誌謝 ii 緒言 iv 中文摘要 v Abstract vii List of figures xii List of tables xiv CHAPTER 1: Background and specific aims 1.1 Avian embryo development 1 1.2 Extraembryonic tissue, yolk sac membrane, and its role in the development 3 1.3 Endodermal epithelial cells 9 1.4 Biofunction of SOAT1 in the mammalian and avian species 13 1.5 The TGF signaling pathway in avian embryonic development 19 1.6 The progress of microRNA sequencing in avian species 21 1.7 Specific aims of the project 24 CHAPTER 2: Material and methods 2.1 MicroRNA (miRNA) sequencing 25 2.2 Prediction of miRNAs targeting genes 27 2.3 Validation of microRNA expressions in YSM tissues of Japanese quail 30 2.4 Cell culture system 2.4.1 EECs culture system 32 2.4.2 HEK293T cell line culture system 37 2.5 Real time PCR for measuring target gene mRNA accumulations 38 2.6 Luciferase plasmid constructions and procedures of luciferase reporter assay 2.6.1 Reagents for molecular cloning preparations 40 2.6.2 Construction of TGFBR1 3’UTR sequence with pmirGLO Dual-Luciferase miRNA target expression vector 41 2.6.2.1 PCR preparation for amplifying the synthetic wild-type 3’UTR or mutated 3’UTR of TGFBR1 42 2.6.2.2 Preparation of Ligation templates 43 2.6.2.3 Ligation and transformation 44 2.6.2.4 Colony confirmation after transformation 45 2.6.2.5 Restriction enzyme digestion preparation (selected plasmid confirmation after amplification) 46 2.6.3 Procedures of luciferase reporter assay 47 2.7 Immunoblotting 49 2.8 Analyses of cellular cholesteryl ester accumulations 51 2.9 Statistical analysis 53 CHAPTER 3: Results 3.1 The candidate miRNAs involving in SOAT1 regulation during embryonic development 54 3.2 The potential functions of selected miRNAs on regulations of SOAT1 and TGFβ signaling pathway 56 3.3 The validations of selected miRNAs pairing ability to the chicken TGFBR1 3’UTR 58 3.4 Verification of interactions between selected miRNAs and the 3’UTR of TGFBR1 60 3.5 SOAT1 is down-regulated by gga-miR-181a-5p and gga-miR-429-3p by modulating TGFBR1 in the TGF signaling pathway 61 CHAPTER 4: Discussions 63 CHAPTER 5: Conclusions 72 CHAPTER 6: References 73 LIST OF FIGURES Figure 1. The workflow of primary EECs collection 32 Figure 2. The map of pUC57 42 Figure 3. The circular map of pmirGLO Dual-Luciferase miRNA target expression vector, and the multiple restriction cutting sites 44 Figure 4. The workflow of wild type sequence of TGFBR1 3’UTR–miRNA pairing activity examination 47 Figure 5. The workflow of mutations on TGFBR1 3’UTR–miRNA pairing activity examination 47 Figure 6. Specific miRNA expressions during embryonic development in YSMs of Japanese quail 99 Figure 7. Target gene expressions after transient transfection for 48 hours using selected miRNAs 101 Figure 8. The mRNA sequence of chicken TGFBR1 102 Figure 9. The insertion of wild-type TGFBR1 3’UTR sequence into pUC57 vector 103 Figure 10. The gel electrophoresis result of synthetic-TGFBR1 3’UTR PCR with restriction enzyme cutting sites (SacI and XhoI) 104 Figure 11. The confirmations of colonies from transformed ligations that with synthetic-TGFBR1 3’UTR constructed in pmirGLO plasmid 105 Figure 12. The results of constructs after restriction enzyme digestions 106 Figure 13. The sequencing result of pmirGLO-syn-TGFBR1-3’UTR 107 Figure 14. The sequencing result of constructed pmirGLO-MU-3’UTR-181a-5p 108 Figure 15. The sequencing result of constructed pmirGLO-MU-3’UTR-429-3p 109 Figure 16. The validations of pairing abilities for selected miRNAs to wild-type chicken TGFBR1 3’UTR 110 Figure 17. Target gene expressions after transient transfection for 48 hours using different concentrations of gga-miR-181a-5p or gga-miR-429-3p 112 Figure 18. The 3’UTR of TGFBR1 was predicted to be one of the direct targets of gga-miR-181a-5p and gga-miR-429-3p 113 Figure 19. The SOAT1 protein expression was regulated by the TGF signaling pathway 115 Figure 20. The conversion of cholesteryl ester was lower in gga-miR-181a-5p and gga-miR-429-3p transfection groups 117 Figure 21. The cholesteryl ester concentration after miRNA transfections for 48 hours and yolk-VLDL stimulation for 24 hours in EECs 119 Figure 22. The possible relationship between miRNAs, TGF signaling pathway and SOAT1 expressions 120 LIST OF TABLES Table 1. The brief summary of miRNA sequencing in avian species 22 Table 2. The list of selected miRNAs 29 Table 3. The miScript primer list 31 Table 4. The Real time-PCR primers 38 Table 5. Primers for TGFBR1 3’UTR amplification and colony confirmation 42 Table 6. The PCR protocol with Phusion polymerase 42 Table 7. The restriction enzyme double digestion protocol for vector and inserts 43 Table 8. The ligation protocol with T4 DNA ligase 44 Table 9. The PCR protocol with Taq polymerase (reaction mix and template) 45 Table 10. The restriction enzyme double digestion protocol for plasmids 46 Table 11. Substrates used for SOAT1 activity detection in previous publications 70 | |
| dc.language.iso | en | |
| dc.title | 日本鵪鶉卵黃囊膜內胚層上皮細胞模式探討小分子miR-181a-5p/ miR-429-3p經TGFβ pathway降低固醇轉醯酶1表現與活性 | zh_TW |
| dc.title | Sterol O-acyltransferase 1 is inhibited by miR-181a-5p and miR-429-3p through the TGFβ pathway in YSM-derived endodermal epithelial cells of Japanese quail | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 劉逸軒,歐柏榮,陳洵一,林原佑 | |
| dc.subject.keyword | 固醇O–醯基轉移?1,乙型轉化生長因子受體1型,卵黃囊膜,內胚層上皮細胞,日本鵪鶉,膽固醇酯, | zh_TW |
| dc.subject.keyword | cholesteryl ester,endodermal epithelial cells,Japanese quail,SOAT1,TGFBR1,YSM, | en |
| dc.relation.page | 121 | |
| dc.identifier.doi | 10.6342/NTU201904449 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2019-12-30 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 動物科學技術學研究所 | zh_TW |
| Appears in Collections: | 動物科學技術學系 | |
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