Plasmid and cell transfection

For overexpression of hsa_circ_0000345, the vector pcDNA3.1 carrying full length of hsa_circ_0000345 was constructed. Empty pcDNA3.1 was used as negative control (NC). To knock down hsa_circ_0000345 or PTEN, small interference RNA (siRNA) specifically targeting hsa_circ_0000345 (Si-hsa_circ_0000345) or PTEN (Si-PTEN), and the corresponding NC (si-NC) were synthesized. The miR-513a-3p mimic, miR-513a-3p inhibitor and the corresponding NC (mimic NC, inhibitor NC) were synthesized. These plasmids were synthesized and purchased from GeneChem (Shanghai, China). Plasmids were transfected into SUNE1 or 6-10B cells using Lipofectamine 2000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) as the protocol of manufacturer. After 48 h of transfection, the transfected cells were collected for further use. The transfection efficiency of hsa_circ_0000345, miR-513a-3p or PTEN was detected by performing quantitative real-time PCR (qRT-PCR).