Bio nc

Biotinylated RNAs, including pre-mature miR-519a-3p wild-type (WT) (bio-pre-miR-519a-3p-WT: 5′- CUGUGACACUCUAGAGGGAAGCGC-3′), mature miR-519a-3p wild type (bio-pre-miR-519a-3p-Mut: 5′-CUGUGGGGCUCUACAAGAAAGCGC-3′), and NC miRNA (bio-NC: 5′-CGAUUGGCUCGAAGCGCGUCAAGA-3′), were purchased from the Invitrogen. For cell transfection, 1 × 10⁶ U87 and U-251 cells were transfected with 45 nM bio-pre-miR-519a-3p-WT, bio-pre-miR-519a-3p-Mut, or bio-NC. Cells were cultivated for 48 h after transfection followed by the preparation of cell lysate samples. The labeled RNAs were then pulled down by using streptavidin agarose magnetic beads Life Technologies (Gaithersburg, MD, USA). After that, total RNAs were isolated and reverse transcription was performed. Finally, RT-qPCR was performed with GAPDH as the internal control to determine the expression levels of circNUP98.

RNA pull-down assay was conducted to further validate the regulatory binding relationship between miR-1179 and CDK2 mRNA. The biotinylated double-stranded RNA of miR-1179 (Bio-miR-1179) and biotinylated negative control RNA (Bio-NC) were designed by GenePharma (Shanghai, China). Whereas the sense sequence of bio-miR-1179 was 5′-AAGCAUUCUUUCAUUGGUUGG-biotin-3′, the antisense sequence of bio-miR-1179 was 5′-CCAACCAAUGAAAGAAUGCUU-3′. 1 × 10⁵ Hela and C-33A cells were cultured in 6-well plates for 1 day, resuspended in 1 ml lysis buffer, and incubated in ice for 20 min. The lysate was centrifuged at 12,000×g for 15 min before the supernatant was collected. The mixture of bio-miR-1179 or Bio-NC and streptavidin-coated magnetic beads (Invitrogen, USA) was added to the supernatant and incubated at 4 °C for 2 h. The pulled-down CDK2 mRNA in the bio-miR-1179 or Bio-NC group was detected by qRT-PCR.

Biotin-labeled molecular probes of miR-1270 (Bio-mimic) or NC (Bio-NC) were obtained from Ribobio. SW1990 and PANC-1 cells were introduced with Bio-mimic or Bio-NC and then lysed by lysis buffer (Invitrogen). RNA complexes in cell lysates were captured by Streptavidin-Dyna beads (Invitrogen). The abundance of NR3C1 in RNA complexes was examined by RT-qPCR.

The assay was performed as described previously [31 (link)]. Biotin-labeled miR-451a (Bio-miR-451a) and negative control (Bio-NC) were synthesized by GenePharma (Shanghai, China). Cells were transfected with Bio-NC and Bio-miR-451a probes for 48 h, following which they were lysed in lysis buffer and incubated with Dynabeads M-280 streptavidin (Invitrogen). Each streptavidin-bound biotin-labeled RNA was eluted using magnetic beads; the eluted RNA was analyzed by qRT-PCR.

Bio‐miR‐486‐5p and Bio‐NC were composed through Thermo Fisher Scientific. Then, the biotinylated miRNA was subjected to cultivation with cell lysates for one night. Afterward, streptavidin magnetic beads were supplemented. In the end, qRT‐PCR was adopted for evaluating expression levels. Each procedure in this experiment was repeated in triplicate.