Anti mir control

The siRNAs specifically targeting SNHG15 (si-SNHG15–1, si-SNHG15–2 and si-SNHG15–3), scrambled negative control (si-control), pcDNA-SNHG15, empty pcDNA vector (vector), miR-141 mimics (miR-141), mimics negative control (miR-control), anti-miR-141 and anti-miR-control were commercially synthesized by Genepharma (Shanghai, China). For transient transfection, U2OS and MG63 cells were plated into six-well plates (2 × 10⁵/well) and routinely maintained for 24 h at 37 °C. Then the cells were transfected with siRNAs, pcDNA-SNHG15, vector, miR-141, miR-control, anti-miR-141, anti-miR-control, si-SNHG15 + anti-miR-141, si-SNHG15 + anti-miR-control, pcDNA-SNHG15 + miR-141, or pcDNA-SNHG15 + miR-control by Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Subsequent experiments were performed at 48 h post transfection. The sequences of si-RNAs, anti-miR-141 are as follows: si-SNHG15–1 (Sense: 5′-CAG GTA GAC CGT GCA CGT AA-3′, Anti-sence:3′-CCT TGA TGC GTT GCC AGC AGA-5′), si-SNHG15–2 (Sense: 5′-CCG TGC GTA AAC GTT TGC CA-3′, Anti-sence: 3′-TGG CGG TAA CGT AAA TGC G-5′), si-SNHG15–3 (Sense: 5′-ACG GTG GCA ACG TGC GTG GCC A-3′, Anti-sence: 3′-GCC TGC AAC GGT GCA AAT GCG-5′), anti-miR-141 (CCA UCU UUA CCA GAC AGU GU UA).

Human coronary artery endothelial cells (hCAECs) were purchased from Lonza (Basel, Switzerland) and were cultured in endothelial cell growth medium consisting of endothelial basal media and EGM-2MV Bullet Kit (Lonza, Basel, Switzerland) at 37 °C in an atmosphere of 95% air and 5% CO₂. Culture media were replaced every 2 days, and the cells were used between passages 6 and 8. When the cells reached 70–80% confluency, a final concentration of 100 nM of synthetic miR mimics (Catalog No. 4464066; Invitrogen, Carlsbad, CA, USA) or anti-miRs (Catalog No. 4464084; Invitrogen, Carlsbad, CA, USA) was transfected into the cells with lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA). At 6 hours post-transfection, the media were replaced, and the cellular lysates were collected 24 hours later for total protein or RNA isolation. A random sequence anti-miR (anti-miR-control, Ambion, Austin, TX, USA) or random sequence pre-miR (pre-miR-control, Ambion, Austin, TX, USA) was used as negative controls, respectively.

MicroRNA-92a inhibition was achieved in vitro by transfecting primary culture podocytes with anti-miR-92a inhibitor using Hiperfect transfection reagent (Qiagen). Anti-miR-Control was used as a control (All from Ambion, 50 nM).