Mir 125b 5p inhibitor

The miR-125b-5p mimic, miR-125b-5p inhibitor, and the relevant negative controls (mimic NC and inhibitor NC) were obtained from Guangzhou RiboBio Co., Ltd. (RiboBio). C2C12 cells were transfected using riboFECT™CP Reagent according to the manufacturer’s instruction. The plasmids for overexpressing were purchased from Genomeditech Biotechnology (Shanghai, China). Cell transfection was performed with the NEOFECT™ DNA transfection reagent according to the manufacturer’s protocol. After transfected for 48 h, the cells were processed for further assays.

The lentiviral vector carrying short hairpin RNA targeting BDNF-AS (sh-BDNF-AS), short hairpin RNA targeting Bcl-2 (sh-Bcl-2), and negative control (sh-NC) were designed and synthesized by GenePharma Co., Ltd. (Shanghai, China). The miR-125a-5p mimics, miR-125b-5p mimics, miR-125a-5p inhibitor, miR-125b-5p inhibitor, and their corresponding negative controls (miR-NC and inhibitor NC) were purchased from Ribobio (Guangzhou, China). Approximately 1.5 × 10⁷ viral particles and 100 pM miRNA mimics or inhibitor were transfected into HEK-293 T, MM.1S, and U266 cells using Lipofectamine 2000 (Thermo Fisher Scientific). After 48 h of transfection, puromycin (Sigma, USA) was added to screen HEK-293 T, MM.1S and U266 cells stably transfected with lentiviruses carrying sh-BDNF-AS, sh-Bcl-2, or sh-NC. The transfection efficiency was confirmed by qRT-PCR. The sequences of oligos used in this study were as follows: 5′-UCCCUGAGACCCUAACUUGUGA-3′ for miR-125b-5p mimic, 5′-TCACAAGTTAGGGTCTCAGGGA-3′ for miR-125b-5p inhibitor, 5′-UCCCUGAGACCCUUUAACCUGUGA-3′ for miR-125a-5p mimic, 5′-UCACAGGUUAAAGGGUCUCAGGGA-3′ for miR-125a-5p inhibitor, 5′-UUCUCCGAACGUGUCACGUTT-3′ for miR-NC, 5′-CAGUACUUUUGUGUAGUACAA-3′ for inhibitor-NC, 5′-GGCTCACCAGTTGTTTGTT-3′ for sh-BDNF-AS, and 5′-UAAGGCUAUGAAGAGA UAC-3′ for sh-NC.

The human nontumorigenic bronchial epithelial cell line BEAS-2B (ATCC, Manassas, VA, USA) was maintained in bronchial epithelial cell growth media (Lonza, Walkersville, MD, USA). The NSCLC cell lines A549, SK-MES-1, and H460 were cultured in RPMI-1640, F-12K (Gibco, Thermo Fisher Scientific, Inc.), and Minimum Essential Medium (Gibco) media, respectively. All the NSCLC cell lines were purchased from ATCC. Additionally, 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin mixed reagent (Gibco) were added to the culture media. The aforementioned cells were maintained at 37°C and grown in a humidified incubator with 5% CO₂ atmosphere.
Three siRNAs against POU6F2-AS2 (si-POU6F2-AS2) expression, NC siRNA (si-NC), E2F3 siRNA (si-E2F3), POU6F2-AS2 overexpression vector (pc-POU6F2-AS2), and E2F3 overexpression vector pcDNA3.1-E2F3 (pc-E2F3), were obtained from GenePharma Company (Shanghai, China). miR-125b-5p mimic, miRNA NC mimic (miR-NC), miR-125b-5p inhibitor, and NC inhibitor were obtained from RiboBio Co., Ltd. (Guangzhou, China). Transient transfection was performed using Lipofectamine 2000 (Invitrogen).