Oe nc

Breast cancer epithelial cell lines (MCF-7, MDA-MB-231, and MDA-10A) and human normal breast epithelial cell line HBL-100 were purchased from the cell bank of Typical Culture Preservation Committee of Chinese Academy of Science. The cells were incubated with DMEM, supplemented with 10% fetal bovine serum (FBS) and penicillin/ streptomycin (Gibco by Life Technologies, Grand Island, NY, USA) at 37°C with 5% CO₂.
Next, MCF-7 cells in the logarithmic growth phase were plated into a 6-well plate (4 × 10⁵ cells/well). When cell confluence reached 70%–80%, the cells were transfected with mimic-negative control (NC), miR-181d-5p mimic, inhibitor-NC, miR-181d-5p inhibitor, oe-NC, oe-HOXA5, sh-NC, or sh-HOXA5, based on the manuals of Lipofectamine 2000 (11668-019; Invitrogen, New York, CA, USA). All of the plasmids were purchased from Shanghai GenePharma (Shanghai, China).

oe-APT1 plasmid (pcDNA3.1-APT1) and its negative control oe-NC (pcDNA3.1-NC), and small interfering RNA si-APT1 and its negative control si-NC were synthesized by RiboBio. Cells in the logarithmic growth phase were transfected with oe-APT1, si-APT1, and their negative controls using Lipofectamine 2000 transfection kit (Invitrogen, Carlsbad, CA, USA).
The osteoblasts were allocated into 8 groups: (1) control (SAMR1 mouse osteoblasts without any treatment); (2) SOP (SAMP6 mouse osteoblasts without any treatment); (3) SOP + oe-NC (SAMP6 mouse osteoblasts transfected with oe-NC for 48 h); (4) SOP + oe-APT1 (SAMP6 mouse osteoblasts transfected with oe-APT1 for 48 h); (5) SOP + si-NC (SAMP6 mouse osteoblasts transfected with si-NC for 48 h); (6) SOP + si-APT1 (SAMP6 mouse osteoblasts transfected with si-APT1 for 48 h); (7) SOP + oe-APT1 + LDN-193189 (SAMP6 mouse osteoblasts cultured in 0.5 μmol/L LDN-193189 medium for 2 d after transfection with oe-APT1 for 48 h); (8) SOP + oe-APT1 + PBS (SAMP6 mouse osteoblasts cultured in an equal concentration of PBS for 2 d after transfection with oe-APT1 for 48 h).

miR-381 mimic, oe-STC2 and their negative controls (NC-mimic and oe-NC) were all obtained from GenePharma (Shanghai, China). Pre-miR-381 sequence was amplified with the forward primer designed as 5ꞌ-CGTGAATGATAGTGAGGAAC-3ꞌ and the reverse primer as 5ꞌ-GTGAACGATTTGCCACACACA-3ꞌ, and then inserted into the PLKO.3G vector. STC2-flag vector was constructed using the pCMV-3tag-3a plasmid for access of STC2 overexpression. Then, pCMV-STC2 (oe-STC2) and pCMV-3tag-3a (oe-NC) were transfected into cells using the Lipofectamine^® 2000 reagent (Invitrogen, Carlsbad, USA), respectively. The RNA concentration was set as 50 nM, and the total RNA and proteins were extracted after 48 h of transfection.

The cells were transfected using Lipofectamine 2000 (11 668 030, Invitrogen Inc., Carlsbad, CA, USA) as per the manufacturer’s instructions. The siRNA targeting DNMT1 mRNA (si-DNMT1), DNMT3A mRNA (si-DNMT3A), DNMT3B mRNA (si-DNMT3B), RARB mRNA (si-RARB) and negative control (si-NC) were acquired from Shanghai GenePharma Co., Ltd. (Shanghai, China). The sequences of the siRNAs are shown in Table 1. The full-length RARB cDNA was subcloned into pcDNA3.1 vector (Invitrogen) for the construction of RARB overexpression (oe-RARB), with the empty vector as a negative control (oe-NC). After seeding and plating in 6-well plates for 1 d, the cells were transfected with the plasmids. After 48 h of transfection, the cells were stored for later use. Transfection efficiency was determined by RT-qPCR.
To suppress the expression of DNMT1, the cells were treated with 10 μM 5-aza-2′-deoxycitidine (5-AzaC) (189 825, Sigma-Aldrich Chemical Company, St Louis, MO, USA) 3 days before transfection. An equal volume of dimethylsulfoxide (DMSO) was applied as a control.