Sirna2

NCI-H1299 or A549 cells (5×10³) were transfected with hsa_circ_0000729 siRNA1 (si-hsa_circ_0000729-1), hsa_circ_0000729 siRNA2 (si-hsa_circ_0000729-2), hsa_circ_0000729 siRNA3 (si-hsa_circ_0000729-3) or negative control (NC; siRNA-ctrl) for 24 h by using Lipofectamine^® 2000 (Invitrogen). Hsa_circ_0000729 siRNA1, siRNA2, siRNA3 and siRNA-ctrl were purchased from Genepharma (Shanghai, China). After 24 h of transfection, cells were harvested of use in the subsequent analysis.
For miR-1281 transfection, NSCLC cells were transfected with miR-1281 agomir, miR-1281 antagomir or NC by using Lipofectamine^® 2000 (Thermo Fisher Scientific). MiR-1281 agomir, antagomir and NC were obtained from Genepharma. After 24 h of transfection, cells were harvested for use in the subsequent analysis.
For hsa_circ_0000729 overexpression, NCI-H1650 cells were transfected with pcDNA3.1 or pcDNA3.1-hsa_circ_0000729 for 24 h by using Lipofectamine^® 2000 (Thermo Fisher Scientific). pcDNA3.1 and pcDNA3.1-hsa_circ_0000729 were obtained from Genepharma.

P2Y2 gene was silenced with small interfering RNA (siRNA) or small hairpin RNA (shRNA). Two P2Y2 siRNA oligonucleotides (siRNA1 and siRNA2) were purchased from Genepharma (Shanghai, China) and the sequences are as follows: P2Y2 siRNA1, 5′‐GUGCUAACAGUUGCCUUGA‐3′ and P2Y2 siRNA2, 5′‐GCCCAAGAGAUGAACAUCU‐3′. A scramble siRNA oligonucleotide was used as negative control siRNA (designated as NC). Cells were transfected with siRNAs using Lipofectamine RNAiMAX Reagent (Invitrogen). An oligonucleotide labeled with fluorescence was applied to directly observe the efficiency of siRNA transfection.
P2Y2 shRNA plasmid as well as a negative control shRNA (NC) was constructed in our lab as described by Li WH et al.12 By using Lipofectamine 2000 (Invitrogen), we transfected MDA‐MB‐231 cells with P2Y2 shRNA or control shRNA, and selected stable clones using G418 (GIBCO).

Two small interfering RNAs (siRNA1 and siRNA2) for CADM1-AS1 silencing and a non-targeting (NT) siRNA were obtained from GenePharma (Shanghai, China). The sequences were as follows: siRNA1, Sense 5ʹ-rGrUrArCrCrUrCrCrUrGrCrCrUrUrUrGrUrCrArArGrCrCAA-3ʹand antisense 5ʹ-rUrUrGrGrCrUrUrGrArCrArArArGrGrCrArGrGrArGrGrUrArCrArA-3ʹ; siRNA2, sense 5ʹ-rGrArCrCrUrArUrCrGrArGrArArCrUrGrArGrArGrCrGrACA-3ʹ and antisense 5ʹ-rUrGrUrCrGrCrUrCrUrCrArGrUrUrCrUrCrGArUrArGrGrUrCrArG-3ʹ. The cells (2×10⁵/ml) seeded in 6-well plates overnight were mixed gently with 5 μg siRNA and 10 μl of Lipofectamine 3000 (Invitrogen, USA) in 250 μl opti-MEM (Gibco) and incubated at 37 °C for 24 h. Then, the medium was replaced with fresh complete medium, and the cells were incubated for a further 24 h.