Mim nc

Human OS cell lines and osteoblast cell line HFOB1.19, available from the American Type Culture Collection (ATCC, Rockville, MD, USA) and the China Center for Type Culture Collection (CCTCC, Wuhan, China), respectively, were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, USA), 100 U/mL penicillin, as well as 100 μg/mL streptomycin (Hyclone, Logan, UT, USA) at 37° C in 5% CO₂. Besides, short hairpin RNA (shRNA) targeting circ_0081001 (sh-circ_0081001), its corresponding negative control (sh-NC), circ_0081001 overexpression plasmid (circ_0081001), and empty vector (negative control, NC) were designed by GeneChem (Shanghai, China). MiRNA-494-3p mimics (miRNA-494-3p mim), miRNA-494-3p inhibitors (miRNA-494-3p inh), and the corresponding negative controls (mim-NC, inh-NC) were available by GenePharma (Shanghai, China). Moreover, Lipofectamine^® 2000 reagent was adopted to transfect the cells. The transfection efficiency, after 36 h, was measured employing quantitative real-time polymerase chain reaction (qRT-PCR).

Human embryonic kidney cells (HEK293T), human BC cell lines (MCF7, SKBR3, MDA-MB-231) and human mammary epithelial cell line MCF-10A were available from the American Type Culture Collection (Rockville, MD, USA). These cells were cultured in Dulbecco’s modified Eagle’s medium (Beyotime, Shanghai, China) with 10% fetal bovine serum (FBS; Beyotime, Shanghai, China), 100 U/mL penicillin and 100 μg/mL streptomycin (Beyotime, Shanghai, China) at 37°C in 5% CO₂. Circ_0048764 overexpression plasmid (pcDNA-circ_0048764), empty vector cDNA (pcDNA-NC), small interfering RNAs (siRNAs) targeting circ_0048764 (si-circ_0048764-1 and si-circ_0048764-2), siRNA negative control (scramble siRNA, si-NC), miR-1296-5p mimics and its control (mim-NC), miR-1296-5p inhibitors and its control (Inh-NC) were produced by GenePharma (Shanghai, China). The above vectors were subsequently transfected into SKBR3 and MCF7 cells by Lipofectamine®3000 (Invitrogen, Carlsbad, CA, USA). 24 h later, quantitative real-time polymerase chain reaction (qRT-PCR) was executed to detect the efficacy.