Anlotinib

Cell viability was measured using Cell Counting Kit-8 (CCK-8; Beyotime Institute of Biotechnology) assays. Control or transfected MCF-7 cells were seeded in 96-well plates at 5×10³ cells/well and incubated at 37°C for 24 h. The cells were the treated with different concentrations of Anlotinib (0, 2, 4 and 6 µM; >99%; Selleck Chemicals) at 37°C for 24 h. A total of 10 µl CCK-8 solution was added to each well for 2 h of culture at 37°C. Cell viability was detected using a microplate reader (Bio-Rad Laboratories, Inc.) at the absorbance of 450 nm.

U2OS, a typical OSA cell line, was purchased from the cell resources center of the Chinese Academy of Medical Sciences (Beijing, China). Five patient-derived cell lines (PDCs) were separated from the clinical specimens obtained from five patients with OSA during surgery as part of normal medical care. The lentivirus particles of pri-miR-596 or Survivin with a mutation of miR-596 targeted sequences located in 3′-UTR were constructed and purchased (Vigene Corporation, Jinan City, Shandong Province, China). The wild-type sequence or sequence with mutated miR-596-binding site of Survivin’s 3ʹUTR (1921–2100) was obtained by chemical synthesis and cloned into pGL4.26 plasmids to construct luciferase reporters by Vigene Corporation. The luciferase containing wild-type sequence of Survivin’s 3ʹUTR with miR-596-binding site was named as Luc, whereas the luciferase containing sequence of Survivin’s 3ʹUTR with mutated miR-596-binding site was named as Luc^Mut. The inhibitor of the miR-596 was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The antitumor agents doxorubicin (Selleck, TX, USA), cisplatin (Selleck), methotrexate (Selleck), or anlotinib (Selleck) were dissolved in DMSO and conserved in an −80°C condition.