73P Generation of miR-21 sponges in lung cancer treatment

Micro-RNAs (miRNA) are post-transcriptional regulatory elements of 17–25 short nucleotides, single-strand and non-coding RNAs. They are essentials for biological processes: such as signal transduction, development, cell growth and cell death. miRNAs may be dysregulated, and about 50% of these alterations are located in cancer-associated genomic regions, leading cells to act in an abnormal or aberrant way. miRNA-21 (miR-21) is usually up-regulated in considerable cancers: breast, colon, glioblastoma, lung, etc. Down-regulation of miR-21 expression by sponges restrains the cell proliferation of non-small cell lung cancer. The aim of this study was to design an efficient method by PCR to generate long DNA sequences containing numerous tandem repeats and used them as sponges for miR-21. Tandemly repeated DNA sequences were generated by PCR from single synthetic oligonucleotide monomers linkers as primers. These primers were hsa-miR-21–5p and reverse complement hsa-miR-21–5p, but with the next modifications: 1) Some nucleotides of theses sequences were changed, because this protects these molecules against endonucleolytic action of protein argonaute 2 (AGO2); 2) Join of two of these sequence by linked nucleotides, because this optimizes binding of miRNAs. Product of PCR was separated by electrophoresis in agarose gel. Primers could pair in different regions, resulting after PCR in fragments variety of sizes from 100 bp to over 1000 bp. Electrophoresis in agarose gel allows choosing the size of the sponge according to the number of repetitions desired. Finally, by other new PCR, selected size could be restricted to generate sponge carrying sticky ends suitable for its insertion in chosen construct without further modification. This grants orientation of the sponge. We developed an effective method for concatenating DNA fragments by PCR. We used this method to obtain sponges for miR-21 of different sizes from 100 bp to over 1000 bp. This allowed us defining length and orientation of sponge for miR-21, leading sponge can be directly used for subcloning or other applications without further treatment. Using sponges for miR-21 has showed the inhibition of miR-21 expression, restraining the cellular proliferation of non-small cell lung cancer.