Anti mirs

For checking the target specificity of miR-15a-3p and miR-302d-3p, we designed LNA modified anti-miRs. The sequence of the oligos are, miR-15a-3p - 5′-TGAGGCAGCACAATATGGCCTG-3′ and miR-302d-3p - 5′-ACACTCAAACATGGAAGCACTTA-3′. The nucleotides marked in bold are LNA modified. After transfection with the 50nM anti-miR (final concentration), qPCR was done for the target transcripts as well as these miRNAs. As an experimental control, we generated a 25-mer scrambled sequence, 5′-CTGCCGGAAGTCGATTGCCCGACGC-3′ which does not have any hit within the human genome. The anti-miRs as well as the scrambled oligo were synthesized from Exiqon.

Anti-miRs were acquired from Exiqon (Denmark). In vivo grade locked nucleic acid-modified Anti-miRs were used. Each anti-miR was dissolved in PBS to create a 2 mg/uL stock which was kept at −20 °C. On the day of injection, each anti-miR was thawed to 4 °C and added to its respective anti-miR cocktail. A total dose of 20 mg/kg for each cocktail was injected. See Supplementary Table 1 for individual inhibitor product number, sequence, and target information.

Pre-miRs (25 nM; Ambion) and anti-miRs (25 nM; Exiqon) were transfected into HUVECs and MDA-MB-231 cells using Dharmafect-4 (Dharmacon Research Inc.) according to the manufacturer's instructions. CCND2, CCND3 and control siRNAs (20 nM) were transfected based on the calcium phosphate transfection method. Transfected HUVECs and MDA-MB-231 cells were plated in EGM2 or complete DMEM, respectively. After a 24-hour transfection, the cells were washed and kept in their media for an additional 48 or 72 hours. Functional assays were performed as described above.