Ghanbari Maryam, Saberfar Esmaeil, Goodarzi Zahra, Lashini Hadi, Ghanbari Sahar, Karamimanesh Mojtaba, Baesi Kazem
Faculty of Advanced Sciences and Technology, Pharmaceutical Sciences Branch, Islamic Azad University (IAUPS), Tehran, Iran.
Researches and Development Department, Bayerpaul Group, Tehran, Iran.
Gastroenterol Hepatol Bed Bench. 2017 Summer;10(3):202-207.
Here, we use miR-122a that exhibits liver-specific expression for developing a post-transcriptional regulative system mediated by microRNAs.
Gene therapy with adenovirus (Ad) vectors that express herpes simplex virus thymidine kinase (HSVtk) and ganciclovir (GCV) have been suggested as a therapeutic strategy to cancer. However, Ad vectors injected into tumors are dispersed into the systemic circulation and transduce liver cells, resulting in severe hepatotoxicity. To be effective, the delivery and expression of suicide genes to cancer treatment ought to be specific to tumor cells, and avoid death of healthy cells. Researchers have demonstrated that expression of transgene could be suppressed in healthy cells with use of vectors that are reactive to microRNA regulation.
We constructed an Ad vector carrying four tandem copies of target sequences of miR-122a that were incorporated into 3'-UTR of HSVtk gene. The expression level of miR-122a was quantified in HepG2 and Huh7 cell lines.
Quantitative RT- PCR analysis demonstrated that Huh7 cells express large amounts of miR-122a compared to HepG2 cells. The viability of Huh7 cells and HepG2 cells after infection by Ad-tk-122aT vector was 83% and 23.5%, respectively. The viability of Huh7 cells was not reduced in the presence of GCV after infection by Ad-tk-122a vector. In contrast, cytotoxicity of HSV-tk/GCV was similar in Huh7 cells and HepG2 cells by Ad-tk vector, with 35.3% and 27% viability, respectively.
Inclusion of the miR-122a target sequences in the HSVtk expression cassette yielded a feasible strategy for reducing cytotoxicity of suicide gene in a liver cell line with high miR-122a expression.
在此，我们使用具有肝脏特异性表达的miR-122a来开发一种由微小RNA介导的转录后调控系统。
用表达单纯疱疹病毒胸苷激酶（HSVtk）和更昔洛韦（GCV）的腺病毒（Ad）载体进行基因治疗已被提议作为一种癌症治疗策略。然而，注入肿瘤的Ad载体分散到体循环中并转导肝细胞，导致严重的肝毒性。为了有效，自杀基因在癌症治疗中的递送和表达应该对肿瘤细胞具有特异性，并避免健康细胞死亡。研究人员已经证明，使用对微小RNA调控有反应的载体可以在健康细胞中抑制转基因的表达。
我们构建了一个携带四个串联拷贝的miR-122a靶序列的Ad载体，这些靶序列被整合到HSVtk基因的3'-UTR中。在HepG2和Huh7细胞系中对miR-122a的表达水平进行了定量分析。
定量逆转录-聚合酶链反应（RT-PCR）分析表明，与HepG2细胞相比，Huh7细胞表达大量的miR-122a。用Ad-tk-122aT载体感染后，Huh7细胞和HepG2细胞的活力分别为83%和23.5%。用Ad-tk-122a载体感染后，在存在GCV的情况下，Huh7细胞的活力没有降低。相比之下，Ad-tk载体在Huh7细胞和HepG2细胞中HSV-tk/GCV的细胞毒性相似，活力分别为35.3%和27%。
在HSVtk表达盒中包含miR-122a靶序列为降低在高表达miR-122a的肝细胞系中自杀基因的细胞毒性提供了一种可行的策略。
