Abstract
We developed a novel miRNA design that significantly improves strand selection within the RISC complex by engineering the 3′ end by adding extra nucleotides. Addition of seven nucleotides at the 3′ ends of the miR or miR* strand resulted in a thermodynamic asymmetry at either of the two ends, which resulted in selective RISC recruitment, as demonstrated by a stem-loop PCR experiment. Such selective recruitment was also corroborated at the protein level by western blot analysis. To investigate the functional effect because of selective recruitment, we performed apoptosis and metastasis studies using human colon carcinoma cells (HCT116) and human osteosarcoma cells (MG63). These experiments indicated that recruitment of the miR strand is responsible for inducing apoptosis and inhibiting the invasiveness of cancer cells. Recruitment of the miR* strand, on the other hand, had the opposite effect. To the best of our knowledge, our strand engineering strategy is the first report of improved strand selection of a desired miRNA strand by RISC without using any chemical modifications or mismatches. We believe that such structural modifications of miR34a could mitigate some of the off-target effects of miRNA therapy and would also allow a better understanding of sequence-specific gene regulation. Such a design could also be adapted to other miRNAs to enhance their therapeutic potential.
Highlights
MicroRNAs belong to the large family of noncoding RNAs that regulate a variety of cellular process employing RNAi
These RNA molecules are formed from long single-stranded RNAs that are transcribed from genomic DNA.[1] miRNA was first identified in C. elegans, where it was found to regulate LinÀ 14 mRNA through the 30 UTR.[2]
We investigated the effect of modified miRNA sequences on apoptosis or necrosis of cancer cells and compared it with miR-34a
Summary
MicroRNAs (miRNAs) belong to the large family of noncoding RNAs that regulate a variety of cellular process employing RNAi. MiRNAs are transcribed by the RNA polymerase II gene and form hairpin loop structures that are processed by Drosha to form shorter doublestranded structures.[3] The shortened 21 to 22 nucleotide binds to mRNA and regulates the translation process or degrades the mRNA.[4] Unlike endogenous small interfering RNA (siRNA), miRNA duplexes have mismatches that allow unwinding of the duplex after binding to a ribonucleoprotein complex called RNAinduced silencing complex (RISC).[5] When the miRNA duplex binds to Argonaute (Ago) within the RISC, only one of the strands is fully incorporated, forming a mature RISC complex, whereas the other strand is discarded and degraded by nucleases in the cytosol.[6] The strand fully bound to RISC is used as a template to find mRNA targets, and most miRNAs have selectivity toward the 30 UTR of the mRNA. Dysregulation of miRNA expression can positively or negatively affect cellular processes
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