Mirna inhibitor nc inhibitor nc

Non-cancerous control cells (GES-1), normal gastric mucosa cell line (RGM-1), and GC cell lines [(HGC-27 (derived from metastatic site: lymph node), AGS (derived from gastric adenocarcinoma), and MKN45 (derived from undifferentiated carcinomas)] were purchased from Procell. GES-1, HGC-27, and MKN45 cells were cultured in RMPI1640 (Procell) supplemented with 10% fetal bovine serum (FBS, Procell), while AGS cells were cultured in Ham’s F-12k (Procell) supplemented with 10% FBS.
Small interfering RNA against LINC00562 (si-lnc) or AP1S3 (si-AP1S3) and their corresponding negative control (si-NC) were provided by Sangon Biotech. MiR-4636 mimic (miR-4636), miRNA mimic NC (miR-NC), miR-4636 inhibitor (inhibitor), and miRNA inhibitor NC (inhibitor-NC) were directly obtained from Ribobio. The experimental cells were subjected to various transfections using Lipofectamine 2000 (Invitrogen) and then incubated at 37°C with 5% CO₂ for 24 h. Next, the cells were collected, and real-time quantitative PCR (RT-qPCR) or Western blotting was conducted to examine transfection efficiency. The sequences of the vectors are summarized in Supplementary Table 1.

The pcDNA circ-FOXO3, miR-543 mimic (5′-AAACAUUCGCGGUGCACUUCUU-3′), miR-543 inhibitor (5′-UACUUAAUGAGAAGUUGCCCGUGUUUUUUUCGCUUUAUUUGUGACGAAACAUUCGCGGUGCACUUCUUUUUCAGUAU-3′), small interfering (si)-LATS1 (5′-GCAATCAGTTAACCGCAAA-3′) and indicated controls, including pcDNA vector, miRNA mimic negative control (mimic NC) (5′-UUUGUACUACACAAAAGUACUG-3′), miRNA inhibitor NC (inhibitor NC) (5′-ACUACUGAGUGACAGUAGA-3′), siRNA NC (si-NC) (5′-GCACAGTTAACCGCATAAA-3′) were obtained from Guangzhou RiboBio Co., Ltd. For cell transfection, cells (1×10⁶) were inoculated into 6-well plates and maintained for 24 h at 37°C, followed by transfection with 100 nM pcDNA circ-FOXO3, miR-543 mimic, miR-543 inhibitor or si-LATS1 using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). After 48 h of transfection, HT29 and HCT116 cells were harvested for subsequent experiments. The transfection efficiency was determined using RT-qPCR and cell fluorescence.
For cell fluorescence, cells were fixed with 4% paraformaldehyde for 10 min at room temperature and incubated with Cy3-labeled miR-543 probe in hybridization buffer at 37°C overnight. The nuclei were stained with DAPI for 5 min at room temperature. Cell fluorescence were captured under the confocal microscope at ×40 magnification (Carl Zeiss AG).