Mirna inhibitor

Cells (2 × 10⁴/well) were incubated in 70% MEM with 10% FBS, 30% Opti-MEM with 15 pM siRNA, or miRNA inhibitor (Applied Biosystems) and 0.25 μl/well of Lipofectamine (Invitrogen) in 96-well plates. For the experiments using inhibitors, cells were treated with either SJ001 (cambogin, 5 μM) [65 (link)] or 10058-F4 (an inhibitor that disrupts the c-MYC-Max interaction) [45 (link)–47 (link)] (Calbiochem) at various concentration as indicated in the figures. At 48 h post-treatment, cell proliferation rates were determined using a MTS assay (Promega).

The miRNA inhibitor and the control for miRNA-455-3p were purchased from Applied Biosystems. The inhibitor and the negative control were mixed with Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) and added for 24 h to PHCjEs at 80% confluence.

Chondrocytes collected from healthy adults (402-05A, Sigma-Aldrich) and from OA patients (402OA-05A, Sigma-Aldrich) were included in this study to match the OA patients and healthy controls included in this study. Cell culture medium was composed of 10% FBS and 90% chondrocyte growth medium (PromoCell). Cell culture conditions were 37 °C, 95% humidity and 5% CO_2. Cells were collected at about 80% confluence to perform transfections. PcDNA 3.1 was used to construct the expression vector of SNHG9 (NCBI Accession: NR_003142.2). Negative control (NC) miRNA (5′-CUGUACGUGCUAGUGCGUGGA-3′) and miR-34a mimic (5′-UGGCAGUGUCUUAGCUGGUUGU-3′) were obtained from Sigma-Aldrich (USA). MiR-34a inhibitor (Cat # HMI0508-5NMOL) and miRNA inhibitor NC (Cat # NCSTUD001) were also purchased from Sigma-Aldrich. Lipofectamine 2000 (Invitrogen) was used to transfect 10⁶ chondrocytes (two types) with 40 nM miR-34a mimic, 40 nM miRNA inhibitor, or 10 nM SNHG9 expression vector following the manufacturer’s instructions. The transfection mixture was incubated for 6 h. Transfection with empty vector, NC inhibitor or NC miRNA was used as the NC group. Untransfected cells were used as the control (C) group. Following experiments were performed at 48 h post-transfection.

MiR-494 mimic, miR-494 inhibitor and the corresponding negative control (mimics NC and inhibitor NC) were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The miRNA mimics, miRNA inhibitor, and the negative control miRNA oligonucleotides were transfected into the HEK293T cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions.

The miRNA mimics, miRNA inhibitors, and siRNAs (GenePharma Co., China) were used for gene overexpression or knockdown. The siRNA sequences are shown in Supplementary Table 1. Before transfection, the BMSCs culture media was removed, and fresh serum-free media (Gibco) was added. For miRNA mimic, miRNA inhibitor, siRNA, or negative control transfection, Lipofectamine 2000 transfection agent (Invitrogen, USA) was used, following the manufacturer’s instructions. Lentivirus transfection [negative control (Vector), WTAP overexpression lentivirus (OE-WTAP), negative control (shNC), and WTAP-knockdown lentivirus (shWTAP)] was performed at a 30–50% cell density. The culture medium was replaced 6–12 h after transfection and every 3 days until the cells reached 80–90% confluency. After 72 h of transfection, stably transfected cell lines were selected using 2 μg/mL puromycin.