Pgl3 vector

The wild-type sequences of CCAT2 and CHK1 that contained binding sites of miR-424 were, respectively, cloned into the pGL3 vector (GenePharma), based on which pGL3-CCAT2 Wt and pGL3-CHK1 Wt were obtained. Correspondingly, miR-424-binding sites were mutated on the above CCAT2 and CHK1 segments, through which pGL3-CCAT2 Mut and pGL3-CHK1 Mut (GenePharma) were constructed. According to instructions of Lipofectamine 2000 kit (Invitrogen, USA), U87 and U251 cells were, respectively, transfected by pGL3-CCAT2 Wt and miR-424 mimic, pGL3-CCAT2 Mut and miR-424 mimic, pGL3-CCAT2 Wt and miR-NC, pGL3-CHK1 Wt and miR-424 mimic, pGL3-CHK1 Mut and miR-424 mimic, as well as pGL3-CHK1 Wt and miR-NC. Four hours of transfection later, the cells were cultivated in a CO₂ incubator (v/v, 5%) at 37°C for 48 hrs. Led by the guidance of dual-luciferase reporter assay kit (Promega, Madison, WI, USA), the luciferase activity of cells was estimated, and its value was equivalent to the ratio of firefly luciferase and renilla luciferase.

The FOSL2-binding site in the promoter of NAMPT was predicted using the JASPAR database (http://jaspar.genereg.net/). Wild-type (wt) and mutant-type (mut) reporter plasmids of NAMPT sequences were amplified by PCR and cloned into the PGL3 vector (GenePharma). Site-directed mutagenesis of the FOSL2 binding site in NAMPT promoter was performed using a site-directed mutagenesis kit (Stratagene, CA, USA). Then, cells were co-transfected with NAMPT-WT, NAMPT-MUT1, NAMPT-MUT2, or NAMPT-MUT1/2 plasmids and Oe-FOSL2 or Oe-NC for 48 h. The luciferase activity was assessed by a dual-luciferase reporter assay system (Promega).

Bioinformatics software Circular RNA Interactome (https://circinteractome.nia.nih.gov/index.html) was used to predict the presence of binding sites for miR-331-3p in hsa_circ_0038646 and GRIK3, and a luciferase reporter assay was performed to validate the association between hsa_circ_0038646, miR-331-3p and GRIK3. SW620 and HCT116 cells were seeded into 24-well plates at the density of 5×10⁴ and co-transfected with wild-type (WT) or mutated (Mut) hsa_circ_0038646, which were inserted into the reporter vector pmirGLO (600 ng; Promega Corporation) and GRIK3 3′-UTR reporter plasmid sequences, which were inserted into the pGL3 vector (600 ng; Shanghai GenePharma, Co., Ltd.) with either miR-331-3p mimics or NC (120 nM), using Lipofectamine^® 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.). After 24 h of transfection, luciferase activity was measured with the Dual Luciferase Reporter Assay System (Promega Corporation) and normalized to Renilla luciferase activity.

The SP1-binding site in the promoter of lncRNA MCF2L-AS1 was predicted using JASPAR database (http://jaspar.genereg.net/). Site-directed mutagenesis of the SP1 binding site in lncRNA MCF2L-AS1 promoter was performed using a site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). Wild-type (wt) and mutant-type (mut) reporter plasmids of lncRNA MCF2L-AS1 sequences were cloned into PGL3 vector (GenePharma). Then, cells were co-transfected with lncRNA MCF2L-AS1-wt or lncRNA MCF2L-AS1-mut plasmids and oe-SP1 or oe-NC by Lipofectamine™ 3000 (Invitrogen). The luciferase activity was examined using a dual-luciferase reporter assay system (Promega).