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研究生:謝善任
研究生(外文):Shan-Ren Xie
論文名稱:MBNL3在卵巢癌細胞中參與的致癌機制
論文名稱(外文):Participation of MBNL3 in the oncogenic mechanism of ovarian cancer cells
指導教授:林庭慧
指導教授(外文):Ting-Hui Lin
口試委員:潘惠錦李娟許仁駿
口試日期:2023-06-29
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:生物醫學科學學系
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:2023
畢業學年度:111
語文別:中文
論文頁數:72
中文關鍵詞:MBNL3卵巢癌細胞凋亡細胞自噬上皮-間質轉化基質金屬蛋白酶
外文關鍵詞:MBNL3Ovarian cancerApoptosisAutophagyEpithelial–mesenchymal transitionMatrix metalloproteinases
DOI:10.6834/csmu202300211
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Muscle blind-like protein 3 ( MBNL3 )是一種調節選擇性剪接的 RNA 結合蛋白,目前已有許多研究指出MBNL3可能與促進癌症發展相關。先前的研究表明,MBNL3 在卵巢癌細胞中表達是增加的,使用專一性弱化MBNL3 的表達減少了卵巢癌細胞增殖、遷移和聚落的形成。本研究旨在探討MBNL3在卵巢癌進展中的致癌機制。 使用表達shRNA ( short hairpin RNA ) 的慢病毒感染卵巢癌 Hey C2 細胞來降低MBNL3之表達。使用反轉錄聚合酶連鎖反應和西方墨點法來確認 sh-MBNL3 慢病毒對 Hey C2 細胞中 MBNL3 的弱化效率。 西方墨點法檢測MBNL3是否參與細胞凋亡、自噬和上皮-間質轉化( Epithelial-mesenchymal transition , EMT )。酵素活性分析用於檢測基質金屬蛋白酶的活性。在本研究中,我們闡明了 MBNL3 對Hey C2 細胞的細胞生長、凋亡、自噬和遷移中的影響。數據表明 MBNL3 在人類卵巢癌中心扮演致癌基因角色,並且 MBNL3 介導的抗細胞凋亡、自噬和 EMT 促進了卵巢癌的進展。弱化 MBNL3 的作用可能有助於作為治療卵巢癌的新策略。
Muscle blind-like protein 3 ( MBNL3 ) is an RNA-binding protein that regulates alternative splicing. Many studies have pointed out that MBNL3 may be related to the promotion of human cancers development. Previous studies showed that MBNL3 expression is increased in ovarian cancer cells, and that specific knockdown of MBNL3 reduces ovarian cancer cell proliferation, migration and the colony formation ability. This study aimed to investigate the oncogenic mechanism of MBNL3 in the progression of ovarian carcinoma. Ovarian cancer Hey C2 cells were infected with lentivirus expressing shRNA (short hairpin RNA ) to reduce the expression of MBNL3.Reverse transcription PCR( RT-PCR ) and Western blots were used to confirm the efficiency of sh-MBNL3 lentivirus on inhibition of MBNL3 expression in Hey C2 cells. Western blots were used to detect whether MBNL3 participated in apoptosis, autophagy and epithelial–mesenchymal transition ( EMT ). Zymography assays were used to examine matrix metalloproteinases ( MMP ) activity. In the present study, we elucidated the effects of MBNL3 on cell growth, apoptosis, autophagy and EMT of Hey C2 cells. Data suggested that MBNL3 plays an oncogene role in human ovarian cancer, and that MBNL3-mediated anti-apoptosis, autophagy, and EMT promote ovarian cancer progression. Inhibiting the effects of MBNL3 may be useful as a new strategy for the treatment of ovarian cancer.
目錄
致謝 i
中文摘要 ii
英文摘要 iii
第一章 序論 (Introduction) 1
Muscle blind-like(MBNL) 蛋白家族 2
MBNL3 參與的生物過程 4
卵巢癌 5
癌症指標 7
細胞凋亡(Apoptosis) 10
細胞自噬(Autophagy) 11
上皮-間質轉化 (EMT) 14
shRNA(short hairpin RNA) 15
研究動機 17
第二章 實驗材料與方法 (Materials and Methods) 19
藥品及試劑 20
實驗方法 26
一、細胞培養 26
二、細胞繼代 27
三、細胞存活檢測(MTT assays) 28
四、RNA萃取與逆轉錄聚合酶連鎖反應(RT-PCR) 29
五、蛋白質萃取與西方墨點法(Western blot)樣品製備及定量分析 31
六、西方墨點法(Western blot) 33
七、免疫螢光染色(Immunostaining) 37
八、酵素活性分析(Zymography assays) 38
九、統計分析 40
第三章 結果 (Results) 41
I. 透過shRNA弱化MBNL3 RNA與蛋白質表現量 42
II. 弱化MBNL3表現對卵巢癌細胞存活的影響 42
III. 弱化MBNL3表現對卵巢癌細胞凋亡路徑蛋白質的影響 43
IV. 弱化MBNL3表現對卵巢癌細胞自噬路徑蛋白質的影響 44
V. 弱化MBNL3表現對卵巢癌細胞中P62的影響 45
VI. 弱化MBNL3表現對上皮-間質轉化蛋白質的影響 46
VII. 弱化MBNL3表現對MMP9蛋白質表現量及活性的影響 47
第四章 討論 (Discussion) 48
圖表 (Figure) 54
FIG(1).Hey C2細胞株轉染sh-MBNL3,對MBNL3表現量的影響 55
FIG(2).弱化MBNL3表現對卵巢癌細胞存活的影響 56
FIG(3).弱化MBNL3表現對卵巢癌細胞凋亡路徑蛋白質的影響 57
FIG(4).弱化MBNL3表現對卵巢癌細胞自噬路徑蛋白質的影響 58
FIG(5).弱化MBNL3表現對卵巢癌細胞中P62的影響 59
FIG(6).弱化MBNL3表現對上皮-間質轉化相關蛋白質的影響 60
FIG(7).弱化MBNL3表現對MMP9蛋白質表現量及活性的影響 61
附圖 62
補充圖表 64
參考文獻 68
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