Mir 326 inhibitor

To silence circ‐TFF1, miR‐326 and TFF1, sh‐circ‐TFF1#1/2/3, miR‐326 inhibitor and sh‐TFF1#1 specifically targeting circ‐TFF1, miR‐326 and TFF1 together with their respective controls (sh‐NC, miRNA NC, sh‐NC) were synthesized and commercially purchased from GenePharma (Shanghai, China). For the downregulation of miR‐326, miR‐326 inhibitor (GenePharma; Shanghai, China) targeting miR‐326 and its negative control were also applied. Transfection of these well‐established plasmids into breast cancer cells was implemented following the instruction of Lipofectamine 2000 reagent (Cat. no: 11668019; Invitrogen; Carlsbad, CA, USA) at a ratio of 1:4.

BMSCs were transfected with miR-326 mimics (5′-CCCCCGTCCCGGAAACACTT-3′) for overexpression the miR-326 level, miRNA mimics control (mimics NC), miR-326 inhibitor (5′-TTACAAAGGCCCTGCCCTGCCCCC-3′) for knockdown the miR-326 level, and miRNA inhibitor control (inhibitor NC) (GenePharma, China). HDAC3 overexpression plasmid pcDNA3.1-HDAC3 (HDAC3 OE) and its negative control plasmid (NC OE) purchased from GenePharma were used to transfect chondrocytes. Cell transfections were performed using the transfection reagent Lipofectamine 2000 (Invitrogen, USA) according to manufacturer's instructions. In brief, cells (3 × 10⁵) were seeded into 6-well plates for cell transfection upon cell fusion of 60~80%. 500 μL Opti-MEM culture medium (Gibco, USA) containing 5 μL Lipofectamine 2000 and 5 μL miR-326 mimics, inhibitors, or controls was added to each well and cultured in an incubator at 37°C for 6 h. The captured cells were treated with 100 nM Trichostatin A or 50 μmol/L AG490 at room temperature for 4 h.