Co-modulation Of MiR-221 And MiR-222 Expression And Function In Glioblastoma By Adenovirus-mediated ShRNAs Produces Distinct Suppression Levels

MicroRNAs (miRNAs) are a set of small single stranded non-coding RNA molecules that serve as important negative gene regulators. Partial complementary pairing between the 3'untranslated region (3'UTR) of the messenger RNA (mRNA) and the 5'seed region of a miRNA is believed to be essential for the post-transcriptional modulation of the target gene's expression. Given the diversity and abundance of target genes, miRNAs appear to functionally interact with various components of cellular signal transduction pathways known as tumor suppressor genes or oncogenes to generate a complex combinatorial network. Examination of those networks for miRNA expression profiles has revealed widespread deregulation of these molecules in diverse cancers, suggesting that miRNAs play critical roles in carcinogenesis.Short hairpin RNAs (shRNAs) are novel modulators that act via RNA interference (RNAi), which contains the pre-designed antisense and sense sequence separated by a loop structure. shRNAs are believed to be processed into their functional form in cells, where the antisense strand functions as a "guide" molecule that leads to an activated form of RISC after the sense strand departure as a "passenger" molecule. shRNAs can function to antagonize aberrantly expressed miRNAs resulting in tumor suppression. Thus, this technique is a potential improvement compared with tumor gene therapy by conventional RNAi or antisense oligonucleotide targeting specific the oncogene. However, in a recent study, shortcomings were reported in the application of lentiviral vectors, and shRNAs are designed to target single miRNAs, not the entire set of aberrant miRNAs and miRNA clusters pertaining to a certain category of cancer.Ciafre et al. and our previous work used a microarray method to show that the expression of the miR-221/222 cluster was strongly upregulated in glioblastoma. Recently, we demonstrated that co-suppression of miR-221 and miR-222 expression by oligonucleotides might inhibit glioblastoma cell proliferation by direct upregulation of the tumor suppressor gene p27kip1 in vivo and in vitro.In the current study, we designed adenovirally-expressed shRNAs that functionally co-repress the expression of miR-221 and miR-222, which are related to glioblastoma, to overcome the low efficiency of former 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 reduced the stability of binding with the miRNA. Various levels of miRNA inhibition 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 extent of 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 suggest that we could produce shRNAs encoded by adenovirus that co-repress multiple tumor-related miRNAs simultaneously, and that the level of repression and the effect on the function of a specific miRNA can be achieved in a semi-quantitative manner. Thus, we unraveled possible concomitant mechanisms of the structural interactions between shRNAs and target miRNAs.