Mir 3619 5p mimic

The fragment containing PVT1, pmirGLO-PVT1-WT, was fused to a pmirGLO vector. pmirGLO-PVT1-Mut is similar to pmirGLO- PVT1-WT, with the mutated binding site of miR-3619-5p. Cos-7 cells (2 × 10⁵/well) were cotransfected with miR-3619-5p mimics or its respective negative control RNA (Guangzhou RiboBio Co. Ltd., Guangzhou, China) in combination with pmirGLO-PVT1-WT or pmirGLO-PVT1-Mut. After 48 h, the Dual Luciferase Assay System (Promega Corporation, Madison, WI, USA) was used to test luciferase activity. PGL3-PVT1-WT: PVT1 5′-flanking region (−1000/0) was fused to pGL-3 luciferase coding sequence. PGL3-PVTI-Mut is equal to pGL-3-PVT1-WT, except that the MKL1-binding CArG box site is mutated from CCTATTTTGC to TTTATTTTAA. Cos-7 cells (2 × 10⁵/well) were cotransfected with MKL1 or its control plasmid (pcDNA3.1-) in combination with pGL3-PVT1-WT or pGL3-PVT1-Mut. After 48 h, the Dual Luciferase Assay System was used to test luciferase activity. The results were expressed as a fold induction relative to the cells transfected with the control after normalization to Renilla activity. For dual luciferase assay results, all columns represent the mean result of three independent experiments, and the error bars represent standard deviation.

MiR-3619-5p mimics, miR-3619-5p inhibitor, and the negative control (control or NC) were purchased from RiboBio. The cells in the six-well plates were transfected with the mimics at 100 nM. The transfection was performed using riboFECTTM CP Reagent (Guangzhou RiboBio Co. Ltd., Guangzhou, China) according to the manufacturer’s instructions.

Plasmids used to knock down LINC00665 (si-LINC00665–1, si-LINC00665–2, and si-LINC00665–3) and the negative control plasmid (si-NC) were purchased from Gene Pharma (Shanghai). miRNA mimics and inhibitors (miR-3619-5p mimic, miR-3619-5p inhibitor, miR-NC mimic, and miR-NC inhibitor) were purchased from RiboBio (Guangzhou). All plasmids were transfected using Lipofectamine 2000 (Invitrogen). The miRNA-binding sites of LINC00665 were predicted using the StarBase 3.0 and miRcode software programs. The wild-type (WT) 3′-untranslated region (UTR) of LINC00665 or CTNNB1 with putative binding sites for miR-3619-5p, or mutant (MUT) 3′-UTRs for each site, were cloned into the psi-CHECK-2 plasmid (Promega) to construct the luciferase reporters WT-LINC00665 and MUT-LINC00665. Luciferase reporter plasmids and the miR-3619-5p or NC mimics were co-transfected into BC cells to analyze the effect of miR-3619-5p on the luciferase activity. After transfection for 48 h, the activities of firefly luciferase (F) and Renilla luciferase (R) were measured using the Dual-Luciferase Reporter Assay System (Promega). The relative luciferase activity of each group was calculated based on the R/F ratio.