Si nc

MiR-103a-3p mimics (5’-AGCAGCAUUGUACAGGGCUAUGA-3’), negative control miRNA (miR-NC: 5’-AUAGCCCUGUACAAUGCUGCUUU-3’), small interfering RNA targeting HMGB1 (siHMGB1: 5’-CUCACCAAGUCUCCUCAAU-3’) and siRNA negative control (siNC: 5’-AUUGAGGAGACUUGGUGAG-3’) were synthesized and purified by GenePharma Co., Ltd (Shanghai, China). These oligonucleotides were cloned into the lentivirus expression vector of Hu6-MCS-CMV-EGFP (GenePharma Co., Ltd). The recombined vector was triple transfected into 80% confluent HEK293T cells with packaging vectors pHelper 1.0 and 2.0 (GeneChem Co., Ltd.) with Lipofectamine 2000 to produce corresponding viral particles. For cell transfection, chondrocytes were divided into 4 different groups, including the miR-NC group, miR-103a-3p group, siNC and siHMGB1 group according the transfected viral particles. They were transfected with scramble miRNA mimics as miRNA (miR-NC), miR-103a-3p mimics, siNC, and siHMGB1, respectively (purchased from Genepharma, Shanghai, China) using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. Forty-eight hours after incubation, the transfection efficiency was observed using a fluorescence microscope (Olympus Co., Ltd., Beijing, China) and miR-103a-3p expression level was evaluated by quantitative real-time PCR.

Human lymphoma cell lines Daudi and Namalwa were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). These two cell types were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. The cells were cultured at 37°C and 5% CO₂. The cell culture reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Cell transfections were conducted using Sinofection regents (Sino Biological, Beijing, China) according to the protocols of manufacturers. The sequence of MCM3AP-AS1-specific siRNA was: 5′-GCTGCTAATGGCAACACTGA-3′, and siNC: 5′-TTCTCCGAACGTGTCACGTTT-3′. The sequence for EIF4E siRNA was: 5′-AAGCAAACCTGCGGCTGATCT-3′; and siNC: 5′-TAAGGCTATGAAGAGATACTT-3′. The miR-15a mimic, inhibitor and their negative controls were synthesized by and purchased from GenePharma (Shanghai, China).

U251 and U87 cells were seeded in 24-well plates at a density of 1 × 10⁵ cells/well and maintained for 24 h at 37°C. Small-interfering RNAs (siRNAs) targeting FGD5-AS1 (si-FGD5-AS1-1 and si-FGD5-AS1-2) and the corresponding negative control (si-NC) were obtained from GenePharma Co., Ltd (Shanghai, China). An overexpression plasmid of miR-103a-3p (miR-103a-3p) and negative control (miR-NC) as well as an miR-103a-3p inhibitor (miR inhibitor) and a negative control (inhibitor-NC) were obtained from GenePharma. U251 and U87 cells were infected with the oligonucleotides stated above using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Cells without transfection were regarded as the blank group. To investigate the interaction between FGD5-AS1-1 and miR-103a-3p, U251 and U87 cells were co-transfected with si-FGD5-AS1 or si-NC and miR inhibitor or inhibitor-NC using Lipofectamine 2000.
pcDNA-TPD52 (TPD52) and negative control (pcDNA-NC) were purchased from GenePharma. To explore whether TPD52 was involved in the role of FGD5-AS1 in GBM, U251 cells were co-transfected with si-FGD5-AS1 or si-NC and pcDNA-NC or pcDNA-TPD52 using Lipofectamine 2000. Cells were analyzed after 48 h of transfection.

The miR-141-3p mimic, mimic negative control (NC), miR-141-3p inhibitor, inhibitor NC, small interfering (si)RNA-circ-LRP6^#1, si-circ-LRP6^#2 and si-NC were designed and synthesized by Shanghai GenePharma Co., Ltd. HOS and SaOS-2 cells (5×10⁶ cells/ml) were transfected as follows: i) The si-NC group transfected with 75 nM si-NC; ii) the si-circ-LRP6^#1 group transfected with 75 nM si-circ-LRP6^#1; iii) the si-circ-LRP6^#2 group transfected with 75 nM si-circ-LRP6^#2; iv) the si-circ-LRP6 + miR-141-3p inhibitor group transfected with 75 nM si-circ-LRP6^#1 + 50 nM miR-141-3p inhibitor; v) the si-circ-LRP6^#1 + pc-HDAC4 group transfected with 75 nM si-circ-LRP6^#1 + 2 µg/µl pcDNA3.1-HDAC4; and vi) the si-circ-LRP6^#1 + pc-HMGB1 group transfected with 75 nM si-circ-LRP6^#1 + 1.8 µg/µl pcDNA3.1-HMGB1. All cells were transfected using Lipofectamine^® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) at room tempera- ture for 48 h and then used for subsequent experimentation. A total of 50 nM miR-141-3p mimic or mimic NC were transfected into HOS and SaOS-2 cells (5×10⁶ cells/ml) using Lipofectamine 3000, as aforementioned, and used in subsequent experiments. The sequences of the transfected siRNAs, miRNA inhibitor, miRNA mimic and the respective controls are presented in Table I.

Three small interfering RNAs (siRNAs) targeting H19 and scramble siRNA (si-NC) were purchased from GenePharma Co. Ltd. (Shanghai, China). The sequences were 5′-CCAACAU CAAAGACACCAUdTdT-3′ for H19-siRNA1, 5′-GCAGGA CAUGACAUGGUCCdTdT-3′ for H19-siRNA2, and 5′-UAAGU CAUUUGCACUGGUUdTdT-3′ for H19-siRNA3. The si-H19 used in this study comprised mixtures of three siRNAs. The sequences for si-NC were 5′-UUCUCCGAACGUGUCACGUTT-3′. miR-130a mimics (miR-130a), miR-130a inhibitor (anti-miR-130a), H19-overexpression plasmid pcDNA-H19 (H19), and corresponding negative controls were all purchased from GenePharma Co. Ltd. for loss- and gain-of-function analyses.
LPS-treated C28/I2 cells were seeded into six-well plates for 24 h of incubation. After that, we transfected oligonucleotides or plasmids into cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. About 48-h post-transfection, cells were collected for following experiments.

Small interfering RNAs (siRNAs) for KCNMB2-AS1 (si-KCNMB2-AS1) and negative control (NC) siRNAs (si-NC) were designed and synthesized by GenePharma Co., Ltd. (Shanghai, China). The si-KCNMB2-AS1#1 sequence was 5ʹ-AACTTTTTATTAGATATCAAAGA-3ʹ; si-KCNMB2-AS1#2 sequence was 5ʹ-ATGAAGATATCTAAAAACAAAGA-3ʹ; si-KCNMB2-AS1#3 sequence was 5ʹ-CTGGATTTCTTGTGAAGAAAACT-3ʹ; and the si-NC sequence was 5ʹ-CACGATAAGACAATGTATTT-3ʹ. An overexpression vector (pcDNA3.1/+) harboring human ρ-associated coiled-coil–containing protein kinase 1 (pcDNA3.1-ROCK1) and empty pcDNA3.1 plasmid were also synthesized by GenePharma Co., Ltd. miR-374a-3p mimic, miR-negative control (miR-NC), miR-374a-3p inhibitor, and NC inhibitor were obtained from RiboBio Co., Ltd. (Guangzhou, China). Cells were seeded into 6-well plates, and Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used for oligonucleotide and plasmid transfections.

siRNA targeting GAS5 (si-GAS5) and siRNA negative control (si-NC) were synthesized by Shanghai GenePharma Co., Ltd. The si-GAS5 sense sequence was: 5′-UCUUCAAUCAUGAAUUCUGAG-3′ and the antisense sequence was: 5′-CAGAAUUCAUGAUUGAAGAAA-3′. The si-NC sense sequence was: 5′-UUCUCCGAACGUGUCACGUTT-3′ and the antisense sequence was: 5′-ACGUGACACGUUCGGAGAATT-3′ (16 (link)). miR-21 inhibitor and inhibitor negative control molecules (inhibitor-NC) were purchased from Guangzhou Ribobio Co., Ltd. The sequence of miR-21 inhibitor was 5′-UCAACAUCAGUCUGAUAAGCUA-3′ and the sequence of inhibitor-NC was 5′-CAGUACUUUUGUAGUACAA-3′. Briefly, 2×10⁵ A549 cells in one well of a 6-well plate were transfected with 50 nM siRNA/si-NC or miR-21 inhibitor/inhibitor-NC mixed in 2 ml Opti-MEM plus 10% FBS using Lipofectamine 2000. Subsequently, 24 h after transfection, the cells were prepared for the subsequent experiments.