Mir nc mimic

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.

The miR-802 mimic, miR-802 inhibitor (anti-miR-802) and their negative control (miR-NC mimic, anti-miR-NC) were achieved by RiboBio (Guangzhou, China). Small interfering RNA (siRNA) sequences targeting circDONSON (si-circDONSON, 5′-AUGUAAAGGUGUUGAUUCCUU-3′), siRNA negative control (si-NC, 5′-UUCUCCGAACGUGUCACGU-3′), pcDNA-BMI1 overexpression vector (pcDNA-BMI1), empty vector (pcDNA-NC), and lentiviral encoding either short hairpin RNA (shRNA)-targeting circDONSON (sh-circDONSON) or a scrambled control sequence (sh-NC) were designed and synthesized by Invitrogen (Carlsbad, CA, USA). Then transfection of these mimics or vectors was carried out using Lipofectamine 2000 (Invitrogen).

The human hsa_circ_0009035 sequence, the hsa_circ_0009035 sequence lacking the mniR-889-3p binding sites, and a nontarget control sequence were synthesized by BGI (Shenzhen, China) and individually inserted into a pCD5-ciR vector (Geneseed, Guangzhou, China) opened with EcoRI and BamHI sites to produce an hsa_circ_0009035 overexpression plasmid, a mutant hsa_circ_0009035 overexpression plasmid (MUT-hsa_circ_0009035), and a negative-control plasmid (pCD5-ciR). Human HOXB7 (accession no. NM_004502.4; synthesized by BGI) was cloned into a pcDNA3.1 vector (Invitrogen, Saint-Aubin, France) with BamHI and XhoI restriction sites to construct HOXB7 overexpressing plasmid, and a nontarget pcDNA3.1 plasmid (pcDNA) was used as the negative-control plasmid. A mature miR-889-3p mimic (5′-UUAAUAUCGGACAACCAUUGU-3′), a mimic negative control (miR-NC mimic; 5′-ACGUGACACGUUCGGAGAATT-3′), an miR-889-3p inhibitor designed for miR-889-3p silencing (anti-miR-889-3p; 5′-ACAAUGGUUGUCCGAUAUUAA-3′), and the scrambled oligonucleotide control (anti-miR-NC; 5′-CAGUACUUUUGUGUAGUACAA-3′) were obtained from Ribobio (Guangzhou, China). HeLa and Siha cells at ∼60% confluence in 24-well plates were transiently transfected with 200 ng of plasmids and 50 nM oligonucleotides using Lipofectamine 3000 as recommended by the manufacturer (Invitrogen). Cells were harvested for further experiments after 48 h.

miR-NC mimic (5′-UUCUCCGAACGUGUCACGU-3′), miR-NC inhibitor (5′-UUGUCCGAACGUGUCACGU-3′), miR-92a-3p mimic (5′-UAUUGCACUUGUCCCGGCCUGU-3′) and miR-92a-3p inhibitor (5′-ACAGGCCGGGACAAGUGCAAUA-3′) were synthesized by RiboBio (Guangzhou, China). miR-92a-3p inhibitor is single-stranded, modified RNA which can tightly bind to endogenous miR-92a-3p and effectively downregulate miR-92a-3p in cells. For transfection, 20 nM miR-NC mimic or miR-NC inhibitor or miR-92a-3p mimic or miR-92a-3p inhibitor was mixed with Lipofectamine 3000 in Opti-MEM and added into the cells in each well of 24-well plate. 48 h after transfection, the transfection efficiency was detected by RT-qPCR.