Gv112 lentivirus vector

The circARHGAP12 overexpression (OV) plasmids and human circARHGAP12 cDNA were synthesized and cloned into the pLC5-ciR vector (Geneseed, Fairfield, CT, USA). To knockdown the expression of circARHGAP12, oligonucleotides encoding short hairpin RNAs (shRNA) specific for circARHGAP12 were designed and synthesized by RiboBio Co., Ltd. (Guangzhou, China) and cloned into the GV112 lentivirus vector (GeneChem). In addition, OS cells were infected by lentivirus with Polybrene (8 mg/mL) and screened by puromycin (2 mg/mL, Sigma-Aldrich) for 2 weeks to obtain stable cell lines. The sequences of shRNA used in present study are listed in Additional file 1: Table S1.

The miR-302e-mimic, miR-302e-inhibitor, oe-CXCL1, short hairpin RNA (shRNA) targeting CXCL1 (sh-CXCL1) and their negative controls (NC) purchased from GeneChem (Shanghai, China) were transfected into CRC cells using Lipofectamine^® 2000 reagent (Invitrogen, Carlsbad, USA) per the manufacturer’s instructions. The oe-CXCL1 designed by GeneChem (Shanghai, China) was cloned into GV112 lentivirus vector (GeneChem, Shanghai, China) for preparation and then used to infect cells with polybrene. 10 μM of AG490 (an inhibitor against JAK-STAT signaling pathway) (Selleck Chemicals, Houston, TX, U.S.A.) or vehicle (DMSO) was transected into cells as indicated. After 48 h, the cells were exposed to 1 mg/ml of puromycin for screening stable colonies.

Full-length PCAT1, SLC7A11, c-Myc, TFAP2C and their negative control cDNA were cloned into the pcDNA3.1 vector (Invitrogen, USA). siRNAs molecules specifically targeting PCAT1, SLC7A11 c-Myc and TFAP2C were designed and synthesized by RiboBio (Guangzhou, China). miRNA mimics and inhibitors of miR-25-3p were also purchased from RiboBio (Guangzhou, China). Lipofectamine 2000 Reagent (Invitrogen, USA) was used to transiently transfect these reagents. In addition, a GV112 lentivirus vector (GeneChem, China) was used to generate short hairpin RNAs (shRNAs) against PCAT1 (labeled as shPCAT1 #1) and a negative control (labeled as shNC). The lentiviral packaging for the PCAT1 expression plasmids and empty control were generated with a lentiviral packaging kit (GeneChem, China). The infected PCa cells were selected with 2 μg/mL puromycin (Sigma-Aldrich, USA) for up to 2 weeks. The siRNA sequences are presented in Table S1.