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研究生:陳佳卉
研究生(外文):Jia-Hui Chen
論文名稱:微型核糖核酸miR-9和miR-221於活化乳癌幹細胞及其乳癌預後角色探討
論文名稱(外文):Enrichment breast cancer stem cells by microRNA miR-9 and miR-22 to predict unfavorable outcome in breast cancer
指導教授:鄭鈞文鄭鈞文引用關係
指導教授(外文):Chun-Wen Cheng
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:生化微生物免疫研究所
學門:生命科學學門
學類:其他生命科學學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:59
中文關鍵詞:乳癌乳癌幹細胞微型核糖核酸
外文關鍵詞:breast cancerbreast cancer stem cellmicroRNA
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癌症幹細胞(CSCs)經由自我更新的能力維持腫瘤生長和轉化為惡性狀態。microRNAs(miRNAs) miR-9和miR-221,已被報導與癌症侵襲性有關聯,經由miRNA過度表現可促進腫瘤細胞幹細胞特性在乳癌的侵襲仍然不明。miR-9和miR-221用於篩選體外的乳腺癌細胞系癌化的側群細胞(SP)。使用雷射顯微擷取系統收集成對的腫瘤細胞和非腫瘤細胞,來自乳腺癌患者的原發性癌組織中為追蹤10年以上。miRNA表現的差異在T(腫瘤)/ N(非腫瘤)比率是使用qRT-PCR定量和分析臨床病理特徵和人類乳癌預後的關係。形態學和腫瘤球群細胞分析數據是根據 miR-9和miR-221的過度表達可以使乳癌幹細胞行程及增加。相反地,癌細胞缺乏幹細胞特性不具有形成球型的潛力,並且在晚期乳癌細胞中抑制了miR-9和miR-221的表現而降低侵襲的能力。在miRNA的過渡表現出在腫瘤組織中歸類為晚期腫瘤分期和淋巴結轉移(LNM)的臨床相關性(P <0.05)。此外,miR-9和miR-221的過度表現出更多數量腫的瘤,高風險的顯著與晚期腫瘤分期(p-trend=0.003)、淋巴結轉移(p-trend=0.028) ,及減少了患者10年腫瘤復發率和整體存活率(log rank P<0.05) 相關。結論miR-9和miR-221的過度表現會形成CSCs和引起乳癌細胞的侵襲和引起,因此開啟潛在早期預測疾病的進展和患者乳癌不好的結果。

Cancer stem cells (CSCs) undergo self-renewal to maintain tumor growth and transform to malignant state. Increased expression of microRNAs (miRNAs), miR-9 and miR-221, has been reported to be associated with cancer aggressiveness, however, the story reside in these miRNA overexpression that endorse tumor cell stemness in outgrowth and invasiveness of breast cancer remains unknown. miR-9 and miR-221 were used to screen in vitro propagation of tumorigenic side-population (SP) of breast cancer cell lines. Paired laser capture microdissected tumor and non-tumor cells were collected from primary cancer tissues of breast cancer patients who were followed up for more than 10 years. Differential expression levels of miRNA in T (tumor)/N (non-tumor) ratio was quantified by qRT-PCR and analyzed their relationship with the clinicopathologic features and outcome of human breast cancer. Morphological and the tumorsphere assay data allows breast CSC formation to be significantly increased upon miR-9 and miR-221 overexpression. On the contrary, cancer cells that lack stem cell properties have limited sphere-forming potential and decreased invasive capability due to inhibition of miR-9 and miR-221 in advanced-stage breast cancer cells. Further, overexpression of both miRNAs showed clinical relevance in tumor tissues categorized as advanced tumor stages and lymph node metastasis (LNM) (p<0.05). In addition, tumors with greater numbers of overexpression of miR-9 and miR-221 were significantly associated with an elevated risk in patients with advanced tumor stage (p-trend=0.003), LNM (p-trend=0.028) and reduced probability of 10-year disease-free survival and overall survival of the patients (log rank P<0.05).Overexpression of miR-9 and miR-221 endow CSCs and given rise to invasiveness of breast cancer cells, thereby opening their potential in earlier prediction of disease progression and an unfavorable outcome in patients with breast cancer.

中文摘要………………………………………………..…..IV
英文摘要………………………………………………..…..V
縮寫檢索…..………………………….………………….….VII
壹、緒論 ( Introduction )
一、 幹細胞與癌幹細胞 ( Stem cell and Cancer stem cell )…....01
二、 乳癌 ( Breast cancer ) ………………………..…………..…02
三、 乳癌幹細胞 ( BCSCs ) …………………………………..…05
四、 微型核醣核酸 (miRNA ) ………………………………..….06
五、 上皮間細胞間質轉化 (EMT) ………………………………08
六、 研究動機……………..…………………………………..…10
貳、實驗材料與方法 ( Materials and Methods )
一、 常用儀器……………….………………………………..…11
二、 材料……………….………………………………..…12
三、 實驗方法……………….………………………………..…17
參、實驗結果 ( Results )
一、 miRNA inhibetor在乳癌細胞株中影響SP cell的表現………..27
二、 經由流式細胞四向分選儀(FACS)分選收集的側群細胞(SP)培養形成乳腺球細胞(mammospheres)的數目…………………...27
三、 利用qRT-PCR確定乳癌幹細胞(BCSCs)的標誌CD133、Nanog、Oct4的表現量………………….………………………………..28
四、 乳癌細胞株轉染miRNAs inhibitor的細胞型態轉變為鵝卵(cobblestone)型態……………………………………………….28
五、 乳癌細胞轉染不同anti-miRNAs減少細胞之侵襲……………29
六、 miRNA mimic影響乳癌細胞株MCF-7生長形成乳腺球體細胞(mammospheres) …………………..……………………………29
七、 傷口癒合試驗(wound healing assay)觀察miRNA mimic影響乳癌細胞株的爬行………………….………………………….….30
八、 miRNA mimic影響之乳癌細胞BCSCs轉移和侵襲的能力….30
九、 女性患者乳腺浸潤性乳管癌(IDC)的臨床特徵……………...31
十、 在女性浸潤性乳管癌( IDC )病患表達不同程度的miRNA在腫瘤細胞相對於非腫瘤細胞………………………………….......31
十一、 女性乳房的浸潤性乳管癌(IDC)在臨床病理特徵與兩個miRNA miR-9及miR-221異常表現之間的相關…………….……...32
十二、 miR-9及miR221過度表現聯合效果( joint effect )在乳房浸潤性0乳管癌( IDC )的腫瘤………………………………….………33
十三、 乳癌患者的不好的存活率(OS)和無病存活率(DFS)有相關性………………….…………………………..34
十四、 Cox regression modle analyses 分析預後共變量(covariates)關於浸潤性乳管癌(IDC)患者之存活率(OS)和無病存活率(DFS) ………………….……………………………………….35

肆、討論 ( Discussion )…………………………………………………36
伍、參考文獻 ( Reference )……………………………………………40
陸、圖表說明 ( Table and Figure )
圖一、 miRNA inhibetor在乳癌細胞株中影響SP cell的表現………..44
圖二、 經由流式細胞四向分選儀(FACS)分選收集的側群細胞(SP)培養形成乳腺球細胞(mammospheres)的數目……………………...45
圖三、利用qRT-PCR確定乳癌幹細胞(BCSCs)的標誌CD133、Nanog、Oct4的表現量………………….……………………….……….46
圖四、乳癌細胞株轉染miRNAs inhibitor的細胞型態轉變為鵝卵(cobblestone)型態……………………………………….………47
圖五、乳癌細胞轉染不同anti-miRNAs減少細胞之侵襲……………48
圖六、 miRNA mimic影響乳癌細胞株MCF-7生長形成乳腺球體細胞(mammospheres) ………………….…………………………….49
圖七、傷口癒合試驗(wound healing assay)觀察miRNA mimic影響乳癌細胞株的………………….………………………….…….……50
圖八、miRNA mimic影響之乳癌細胞BCSCs轉移和侵襲的能力….51

圖九、在女性浸潤性乳管癌( IDC )病患表達不同程度的miRNA在腫瘤細胞相對於非腫瘤細胞………………………………….......52
圖十、miR-9和miR-221與乳癌患者的不好的存活率(OS)和無病存活率(DFS)有相關性………………….……………………………53
表一、 女性患者乳腺浸潤性乳管癌(IDC)的臨床特徵……………...55
表二、 女性乳房的浸潤性乳管癌(IDC)在臨床病理特徵與兩個miRNA miR-9及miR-221異常表現之間的相關…………….……...57
表三、miR-9及miR221過度表現聯合效果( joint effect )在乳房浸潤性乳管癌( IDC )的腫瘤…………………………………………..58
表四、 Cox regression modle analyses 分析預後共變量(covariates)關於
潤性乳管癌(IDC)患者之存活率(OS)和無病存活率(DFS) ………………….…………………………………59


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