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研究生:謝尚潔
研究生(外文):Shang-Chieh Hsieh
論文名稱:沙門氏菌誘導之微核醣核酸376a透過抑制細胞週期進而促進沙門氏菌之增生
論文名稱(外文):Salmonella-induced miR-376a Accelerates Salmonella Replication via Arrest of Host Cell Cycle
指導教授:俞松良俞松良引用關係
指導教授(外文):Sung-Liang Yu
口試日期:2017-06-20
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:醫學檢驗暨生物技術學研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:英文
論文頁數:64
中文關鍵詞:鼠傷寒血清型腸道沙門氏菌微核醣核酸376aHT-29細胞細胞週期G2/M週期延遲
外文關鍵詞:Salmonella enterica serovar TyphimuriummiR-376aHT-29cell cycleG2/M arrest
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許多病原菌透過干擾素主的細胞週期延緩細胞的分裂和上皮組織的更新,使得菌株在上皮細胞的附著時間延長,進而增加感染成功的機會。此外,病原菌也會透過干擾素主的微核醣核酸表現量以抵抗宿主的防禦機制。研究已知鼠傷寒血清型腸道沙門氏菌會促進宿主細胞週期G1/S的轉變,同時也會使宿主細胞停留在G2/M 週期,細胞週期的干擾能促進沙門氏菌在宿主細胞內的複製。本篇研究發現沙門氏菌感染人類結直腸腺癌細胞 HT-29後8小時,透過誘導微核醣核酸376a的表現量增加,造成宿主細胞停留在G2/M 週期。如果抑制HT-29細胞的微核醣核酸376a表現量,則細胞感染後 16 和 24 小時細胞內的沙門氏菌數量有顯著減少。我們分析微核醣核酸376a 下游的基因發現ANGEL2 和 PPP1R10參與細胞週期的調控,且實驗證實微核醣核酸376a 直接抑制這兩個基因的表現。細胞受沙門氏菌感染時,抑制微核醣核酸376a的表現量能減少G2/M週期延遲的細胞數,但此時同時抑制ANGEL2 或 PPP1R10 可以回復G2/M週期延遲的細胞數。這些結果發現沙門氏菌感染會誘導微核醣核酸376a的表現,藉由微核醣核酸376a抑制其目標基因造成宿主細胞的G2/M 細胞週期延遲。
Many bacterial pathogens can interfere with the cell cycle of infected cells to promotes their colonization. Bacterial pathogens also can interfere with the expression of microRNA(miRNA) in order to overcome host defenses. Salmonella enterica serovar Typhimurium promote G1/S transition and induce G2/M arrest to increase intracellular bacterial replication. We find that intracellular Salmonella induces the G2/M phase arrest of epithelial cells after 8 hours post-infection (h.p.i.), and it is dependent on the Salmonella-induced miR-376a. Inhibition of miR-376a in HT-29 cells results in decrease in intracellular replication of Salmonella at 16 and 24 h.p.i. Analysis of miR-376a targets reveals that the expression of two genes, ANGEL2 and PPP1R10, is inhibited by miR-376a. Inhibition of miR-376a reduces Salmonella-induced G2/M phase arrest, and silencing of ANGEL2 or PPP1R10 can reverse the inhibitory effect of the miR-376a antagomiR on G2/M phase arrest. Collectively, these findings suggest that upregulation of miR-376a upon Salmonella infection induces G2/M phase arrest by miR-376a targets inhibition.
Introduction
1. Salmonella Species and Subspecies..………………………..…………….…………1
2. Salmonellosis and Infection…………...…………...……………….………………..3
3. Host Defenses against Bacterial Pathogens………………………………...………...4
4. Bacteria Pathogen against Rapid Epithelial Turnover……………………….……….6
5. MicroRNAs………………………………………………………………………….8
6. Modulation of Host miRNAs by Intracellular Bacterial Pathogens………..……..…..9



Results
1. The Expression Profile of miR-376a during Salmonella Infection……………… 11
2. The G2/M Cell Cycle Arrest Induced by Salmonella Infection Is contributed by miR-376a ...………………………….…………………………………….…………......12
3. ANGEL2 and PPP1R10 Are Targets of miR-376a…………………….……………15
4. ANGEL2 and PPP1R10 Involved in miR-376a-Induced Cell Cycle arrest…….…...17

Discussion………………………………………………………………...…………....20







Figures
Figure 1. The Expression Profile of miR-376a-1 and miR-376a-2 during Salmonella Infection……………………………………………………………...………25
Figure 2. The Expression Profile of miR-376a during Salmonella Infection ……...........26
Figure 3. Specific Inhibition of miR-376a Prevent Salmonella-Induced G2/M Cell Cycle Arrest …..…………...………………………...……………………..…........27
Figure 4. The Expression Profile of miR-376a in AntagomiR-376a Transfected HT-29 Cells during Salmonella Infection……………...…………………….……....29
Figure 5. Specific Overexpression of miR-376a Increase Intracellular Salmonella-Replication.......................................................................................................30
Figure 6. Ectopic Expression of miR-376a Does Not Affect the Cell numbers of Salmonella-infected Cells…………………………………………………....31
Figure 7. Expression of miR-376a Does not Affect PARP1 Cleavage…………..……...33
Figure 8. Gene Ontology Profiles miR376a-Targeted Candidates during Salmonella Infection.……………………………………………………………….…….34
Figure 9. ANGEL2 and PPP1R10 Are a Target of Salmonella-Induced miR-376a…….35
Figure 10 The Expression of miR-376a in shRNA Transfected HT-29 cells………..…37
Figure 11 Reduction of G2/M Cells, which was Induced by antagomiR-376a, was Partially Rescued by ANGEL2 and PPP1R10 shRNAs……………………..38
Figure 12 Model for the Regulatory Role of miR-376a in Salmonella Infection……...39

Table
Table 1. Primers for Plasmid Constructions………………………………………..…..40

Materials and Methods…………………………………………………………...…41

References………………………………………………………………..…………51
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