Pre mir 21

To knockdown HIF-1α expression, the synthesized siRNA oligonucleotides against HIF-1α were transfected in cells, along with the scrambled oligonucleotides. After transfection for 48 h, the cells were harvested and subjected to western blotting to analyze HIF-1α expression. β-actin was used as an internal control. For miR-21 knockdown, a miR-21 inhibitor (LNA-anti-miR-21; Exiqon, Woburn, MA, USA) was added to the culture medium at a final concentration of 30 nM. For miR-21 upregulation, pre-miR-21 (Ambion, Grand Island, NY, USA) was added directly to the complexes and used at a final concentration of 30 nM. PDCD4 and Fas-L gene upregulation was performed by Ad-PDCD4 (30 MOI) and Ad-Fas-L (30 MOI), respectively. The transfection medium was replaced with regular culture medium 4 h post-transfection. Vehicle control, oligo controls for anti-miR-21 (Exiqon) and pre-miR-21 (Ambion), and adenovirus control (Ad-GFP) were applied.

For transient over-expression/downregulation of miRs, cells at 50% confluence were transfected using Oligofectamine (Invitrogen, Milan, Italy) and 100nM of pre-miR-21, pre-miR-143-3p, pre-miR-378e, or scrambled pre-miR; or 200nM of anti-miR-21, anti-miR-143-3p, anti-miR-378e, or scrambled anti-miR (Ambion®, Life Technologies). For miR over-expression, exosomes isolated with ExoQuick-TC™ solution were transfected using Exo-Fect™ Exosome Transfection Reagent (SBI, System Biosciences) and 130nM of pre-miR-21, pre-miR-143-3p, pre-miR-378e, or scrambled pre-miR.

Cells were plated on 60 mm² plates at a density of 1 × 10⁶ cells per plate. When the cells were 70–80% confluent, they were transfected with pre-miR-21 or control pre-miR (Ambion, Austin, TX) using TransIT TKO transfection reagent (Mirus Bio, Madison, WI). Twenty-four hours post-transfection, cells were collected for use in all assays. Tissue inhibitor of metalloproteinase 3 (TIMP3)-specific small-interfering RNA (siRNA) and negative control constructs were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). The sequences for each oligo are as follows: