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.
