中國醫藥大學機構典藏 China Medical University Repository, Taiwan:Item 310903500/41370
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    题名: 發展溶瘤腺病毒雙重調控機制共同標的前列腺癌細胞與支持骨基質
    Cotargeting Prostate Cancer and Supporting Bone Stroma Using Dual Controlled Oncolytic Adenovirus
    作者: 吳怡蕙
    贡献者: 癌症生物學研究所碩士班
    关键词: Ad-hOC-E1;前列腺癌;人類 osteocalsin 啟動子;microRNA 治療 Ad-hOC-E1;prostate cancer;hOC promoter;microRNA therapy
    日期: 2011-07-29
    上传时间: 2011-10-17 16:54:17 (UTC+8)
    出版者: 中國醫藥大學
    摘要: 我們實驗室先前開發的條件複製型腺病毒載體(conditional replicating adenoviral vector, CRAd),Ad-hOC-E1共同標的骨轉移性前列腺癌細胞(PCA)和骨基質。由於腫瘤微環境的重要性,支持腫瘤生長和轉移,Ad-hOC-E1是優於目前臨床使用的單一標的CRAds用於治療前列腺癌骨轉移。據了解,microRNA 抑制基因表達的機制主要調控在轉錄後層面 (post-transcriptional level),其中發現在許多癌症的發展中,microRNA (miRNA) 表現量會下降。我們認為將microRNA調控納入到Ad-hOC-E1,可限制其毒性於正常的組織而不影響癌症相關基質表現。要選擇合適的microRNA,我們進行 microRNA microarray比較正常骨基質細胞和前列腺癌相關骨基質細胞。通過 RT- qPCR,我們證實了miR-195和miR-199a在前列腺癌和癌症都相關基質表現量較低,但是過度表達於正常基質。為了測試是否miR-195和miR-199a的target sequence能夠抑制在正常基質基因的表達,建立了microRNA的調節螢光素酶通過插入合成的miR-195T與miR-199aT和miR-scrambleT到3'-UTR。我們的研究結果表明,miR-199aT顯著抑制正常基質的螢光素酶表達,但不影響和前列腺癌相關的基質。同樣,將miR-195T與miR-199aT崁入腫瘤特異性特設啟動子(tumor-specific hOC-promoter) ,螢光素酶在正常基質基因的表達也減少。另外,將miR-199aT崁入溶瘤腺病毒,使用雙向腫瘤特異性特設啟動子,螢光素酶在正常基質基因的表達也減少。這些結果表明,miR-199aT調節的溶瘤腺病毒,雙重控制腫瘤特異性啟動子和microRNA調節可以提供一個安全的腫瘤靶向方法。
    Our laboratory previously developed a conditional replicating adenoviral vector (CRAd), Ad-hOC-E1 to co-target bone metastatic prostate cancer cells (PCa) and bone stroma. Owing to the importance of tumor microenvironment in supporting cancer growth and metastasis, Ad-hOC-E1 is superior to currently clinically used signal-targeting CRAds for the treatment of bone metastasis PCa. It has been known that microRNAs inhibit gene expression at post-transcriptional level and many of them are dyregulated during cancer progression. We hypothesized that incorporating microRNA regulation into Ad-hOC-E1 could restrict toxicity of microRNAs to cancer-associated stroma but not normal tissues. To select the appropriate microRNAs, we performed microRNA microarray using normal and PCa-associated bone stromal cell lines. By RT-qPCR, we confirmed that miR-195 and miR-199a were down-regulated in both PCa and cancer-associated stroma but over-expressed in normal stroma. To test whether the miR-195 target sequence (miR-195T) or miR-199a target sequence (mir-199aT) is able to suppress the expression of transgene in normal stroma, we constructed microRNA-regulated luciferase reporter by insertion of synthetic miR-195T, miR-199aT and miR-scrambleT into the 3’-UTR of pMIR. Our results demonstrated that miR-199aT significantly suppressed luciferase expression in normal stroma but not in Pca and Pca-associated stroma in comparison to mir-scrambleT. Similarly, incorporating miR-199aT into luciferase expression cassette driven by tumor-specific hOC-promoter also decreased luciferase expression in normal cells. Further, inserting miR-199aT into oncolytic adenovirus driven by bidirectional hOC-promoter could also de-target virus replication in normal cells. These results suggested that miR-199aT-regulated oncolytic adenovirus dual control by tumor-specific promoter and microRNA regulation could provide a safe tumor-targeting approach.
    显示于类别:[癌症生物學研究所] 博碩士論文

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