Adenovirus-mediated shRNAs for co-repression of miR-221 and miR-222 expression and function in glioblastoma cells
Adenovirus-mediated shRNAs for co-repression of miR-221 and miR-222 expression and function in glioblastoma cells
复制标题
腺病毒介导的 shRNA 可共同抑制胶质母细胞瘤细胞中 miR-221 和 miR-222 的表达和功能。
DOI：
10.3892/or_00001047
复制
发表时间：
2011-01-01
期刊：
影响因子：
4.2
通讯作者：
Kang, Chunsheng
中科院分区：
文献类型：
--
作者：
Wang, Xuan;Han, Lei;Kang, Chunsheng
异常表达的miRNA与细胞信号转导通路网络内的癌基因和/或肿瘤抑制基因的调节相关，从而促进癌发生。miRNA的功能可以被拮抗，因此代表了一种用于综合癌症治疗的新的抗肿瘤方法。在这项研究中，我们设计了腺病毒表达的shRNA，功能上共抑制与胶质母细胞瘤相关的miR-221和miR-222的表达，以克服基因治疗的低效率。此外，我们产生了新的shRNA，其3'末端在与靶miRNA的5种子区互补的区域中突变，以降低与miRNA结合的稳定性。实现了各种miRNA抑制水平：经典shRNA产生了miRNA水平的最大降低，其次是突变的shRNA和空白对照，如通过qRT-PCR测定的。这些结果通过miR-221/222的验证靶点p27（kip 1）的蛋白表达、对细胞周期停滞在G（1）期的影响以及对细胞凋亡的影响得到证实。这些结果表明，我们可以产生由腺病毒编码的shRNA，其同时共抑制多种肿瘤相关的miRNA，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。
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 p27(kip1), the validated target of miR-221/222, the effect on cell cycle arrest in G(1) 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.