Mir 140 5p mimic

For 24 h miRNA mimic transfection, mHypoE-46 and mHypoE-41 cells were grown to 70–80% confluency in 60 mm tissue culture plates. mirVana miRNA mimic negative control #1 (ID: 4464061), miR-29b-1-5p mimic (ID: MC12431), miR-140-5p mimic (ID: MC10205), miR-143-3p mimic (ID: MC10883), let-7f-1-3p (ID: MC13032), and let-7b-3p (ID: MC12489) were purchased from Thermo Fisher Scientific (Burlington, ON, Canada). For transient transfection, 25 nM mimic or negative control solutions were complexed with Dharmafect 3 Transfection Reagent (Dharmacon, Cedarlane, Burlington, ON, Canada) for 20 min at room temperature in serum- and antibiotic-free plain DMEM with 4500 mg/L glucose (Millipore Sigma). The complexed transfection mixture was then diluted 1:10 with antibiotic-free DMEM with 4500 mg/L glucose (Millipore Sigma) supplemented with 2% FBS. Cells were washed with PBS, and the transfection media was added to the cells for 24 h.

A normal cell line hFOB 1.19 [CBP60724; Culture Medium: Dulbecco's modified Eagle's medium (DMEM): F12 + 0.3 mg·mL⁻¹ G418 + 10% FBS) was cultured with 5% CO₂ at 34 °C as described previously (Bozycki et al., 2018) and five cell lines related to osteosarcoma: U2OS (CBP60238; Culture Medium: McCoy's 5a + 10% FBS), SaOS‐2 (CBP60742; Culture Medium: McCoy's 5a + 15% FBS), MG63 (CBP60233; Culture Medium: MEM + 10% FBS), HOS (CBP60787; Culture Medium: RPMI‐1640 Medium + 10% FBS), and SJSA1 (CBP60236; Culture Medium: RPMI‐1640 + 10% FBS), which were purchased from Cobioer Biotechnology Co., Ltd. (Nanjing, China) and cultured with 5% CO₂ at 37 °C. The medium was renewed every day.
Osteosarcoma cells were plated into 6‐well plates (3 × 10⁵ cells/well). Upon attaining 50% cell confluence, short hairpin RNA (sh)‐negative control (NC), sh‐PGM5‐AS1‐1, sh‐PGM5‐AS1‐2, overexpression (oe)‐NC, oe‐PGM5‐AS1, inhibitor NC, miR‐140‐5p inhibitor, mimic NC, miR‐140‐5p mimic, or sh‐FBN1 was delivered into the cells following the procedures in the Lipofectamine 2000 kit (11668‐019; Invitrogen, Carlsbad, CA, USA). The mimic NC, miR‐140‐5p mimic, inhibitor NC, and miR‐140‐5p inhibitor, and plasmids were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). Table 1 displays the sequences of shRNAs.