Mircury lna microrna target site blockers

Lipofectamine RNAiMAX (Life Technologies) was used to transfect HAECs with a LNA-miR-103 inhibitor (50 nM, miRCURY LNA microRNA Inhibitors; Exiqon), a miR-103 mimic (15 nM, mirVana mimics; Life Technologies), Dicer GapmeRs (10 nM, LNA GapmeRs; Exiqon), miR-103-KLF4 target site blockers (50 nM miRCURY LNA microRNA Target Site Blockers; Exiqon), premade KLF4 mRNA (2 μg, mRNAExpress Human KLF4 Transcript; BioCat GmbH) or scrambled controls. Total RNA was isolated after 24 or 48 h using the RNeasy Mini Kit (Qiagen) or mirVana Isolation Kit (Life Technologies). HAECs were transfected with a small interfering RNA (siRNA) against KLF4 or a non-targeting siRNA (1 μM Accell siRNA in Accell Delivery Cell Culture Medium; Thermo Scientific) for 72 h. Additional treatment with the LNA-miR-103 inhibitor was performed as described above.

Cell were transfected with Pre-miR™ miRNA Precursors for hsa-miR-493-3p and negative control #1 (Ambion, Thermo Fisher Scientific, Waltham, MA, USA), Anti-miR™ miRNA Inhibitor for hsa-miR-493-3p (Ambion) and with miRCURY LNA™ microRNA Target Site Blockers (Exiqon, Denmark) using Hiperfect (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol for reverse transfection. The sequence of MAD2-TSB was 5′-ATGAAGGTCAAAAGGAGCTA-3′ and for the control-TSB 5′-AGAGCTCCCTTCAATCCAAA-3′. All pre-miRNAs and target site blockers were used at 50nM final concentration. In luciferase assays, pre-miRNAs and plasmids (50 ng) were forward transfected with Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific). The E2F1 siRNAs used were siE2F1_1: CUCACUGAAUCUGACCACC and siE2F1_2: CAGAUCUCCCUUAAGAGCA at 20nM final concentration.

A synthetic scrambled miR mimic and a miR‐34a mimic (catalog numbers SI03650318 and MSY0000542, respectively; Qiagen) were transfected into primary mouse lung fibroblasts with HiPerFect (Qiagen) or MLg cells with Lipofectamine^® 2000 or 3000, as per manufacturer's instructions. Locked nucleic acid (LNA) oligonucleotides (purchased from Exiqon) included a scrambled (inert) sequence (5′‐ACGTCTATACGCCCA‐3′); an antimiR directed against miR‐34a (5′‐AGCTAAGACACTGCC‐3′) and miRCURY LNA™ microRNA Target Site Blockers (herein referred to as target site blockers) directed to target the interaction between the two miR‐34a binding sites in the mouse Pdgfra 3′‐UTR and miR‐34a: 5′‐TTGGCAGTATTCTCCA‐3′ (TSB1) and 5′‐AGGCAGTGATACAGCT‐3′ (TSB2) (see Fig 3A). In vitro, synthetic oligonucleotides were transfected into MLg cells with Lipofectamine^® 2000. When combined, synthetic microRNA mimics and LNA target site blockers were applied together as a cocktail, at a final cumulative concentration of 160 nM (Fig 4B). In vivo, both target site blockers (applied as a cocktail of a 1:1 mixture of TSB1 and TSB2) and a scrambled or miR‐34a‐specific antimiR were all applied by intraperitoneal injection at a dose of 10 mg/kg at P1 and P3, in ddH₂O.