Pcdna3.1 sox9

To overexpress miR-431-5p and SOX9, transfection of the miR-431-5p mimic (100 pmol; Shanghai GenePharma Co., Ltd.) and pcDNA3.1-SOX9 (4 μg; Sangon Biotech Co., Ltd.) was conducted with Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and the miRNA mimic control (100 pmol; miR-NC; Shanghai GenePharma Co., Ltd.) and pcDNA3.1 (4 μg;) were used as controls. All transfections were performed at room temperature. The transfection duration was 8 h, after which the culture medium was replaced with fresh culture medium.
Small interfering RNAs (siRNAs) targeting LINC01132 (si-LINC01132; 100 pmol) and the miR-431-5p inhibitor were prepared by Shanghai GenePharma Co., Ltd. and used to knock down LINC01132 and miR-431-5p. The negative control (NC) siRNA (si-NC; 100 pmol) and NC inhibitor functioned as the controls for si-LINC01132 and the miR-431-5p inhibitor, respectively. The miR-431-5p mimic sequence was 5′-ACGUACUGCCGGACGUUCUGU-3′ and the miR-NC sequence was 5′-UUGUACUACACAAAAGUACUG-3′. The miR-431-5p inhibitor sequence was 5′-UGCAUGACGGCCUGCAAGACA-3′ and the NC inhibitor sequence was 5′-ACUACUGAGUGACAGUAGA-3′. The si-LINC01132#1 sequence was 5′-AGGAGATAAAAATTTTAAATTAC-3′; the si-LINC01132#2 sequence was 5′-TTCTGTTTTTTGTTTTTTTAAGA-3′; and the si-NC sequence was 5′-CACGATAAGACAATGTAT TT-3′.