Department of Neurosurgery, Tianjin Medical University General Hospital, Tianjin, P.R. China.
Oncol Rep. 2011 Jan;25(1):97-105.
Aberrantly expressed miRNAs are linked to the regulation of oncogenes and/or tumor suppression genes within the cell signal transduction pathway network, thereby contributing to carcinogenesis. miRNA function can be antagonized, thus representing a novel anti-tumor approach for integrated cancer therapy. In this study, we designed adenovirally-expressed shRNAs that functionally co-repressed the expression of miR-221 and miR-222, which are related to glioblastoma, to overcome the low efficiency of gene therapy. In addition, we generated novel shRNAs whose 3' ends were mutated in the region complementary to the target miRNA's 5' seed region to reduce the stability of binding with the miRNA. Various inhibition levels of miRNA were achieved: classic shRNAs yielded the greatest reduction in miRNA levels, followed by mutated shRNAs and the blank control, as determined by qRT-PCR. These results were confirmed by the protein expression of p27kip1, the validated target of miR-221/222, the effect on cell cycle arrest in G1 phase, and the impact on cell apoptosis. These results suggested that we could produce shRNAs encoded by adenovirus that co-repressed multiple tumor-related miRNAs simultaneously, and that the level of repression and the effect on the function of a specific miRNA could be achieved in a semi-quantitative manner.
异常表达的 miRNA 与细胞信号转导通路网络中的癌基因和/或肿瘤抑制基因的调节有关，从而促进了癌症的发生。miRNA 的功能可以被拮抗，因此代表了一种新的用于综合癌症治疗的抗肿瘤方法。在本研究中，我们设计了腺病毒表达的 shRNA，以功能性地共同抑制与神经胶质瘤相关的 miR-221 和 miR-222 的表达，以克服基因治疗的低效率。此外，我们生成了新的 shRNA，其 3' 端在与 miRNA 的 5' 种子区互补的区域发生突变，以降低与 miRNA 结合的稳定性。实现了各种 miRNA 抑制水平：通过 qRT-PCR 确定经典 shRNA 产生的 miRNA 水平降低最大，其次是突变 shRNA 和空白对照。通过 p27kip1 的蛋白表达、miR-221/222 的验证靶标、对 G1 期细胞周期阻滞的影响以及对细胞凋亡的影响证实了这一结果。这些结果表明，我们可以产生同时共同抑制多种肿瘤相关 miRNA 的腺病毒编码 shRNA，并且可以以半定量的方式实现抑制水平和对特定 miRNA 功能的影响。
