Mir 543 mimic

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).

Small interfering RNA (siRNA) specifically targeting ST7-AS1 (si-ST7-AS1#1, si-ST7-AS2, and si-ST7-AS1#3), transient receptor potential melastatin 7 (TRPM7) siRNA (si-TRPM7), and negative control siRNA (si-NC) were produced by Generay Biotech (Shanghai, China). miR-543 mimic, miRNA NC mimic (miR-NC), miR-543 inhibitor, and NC inhibitor were obtained from RiboBio Technology (Guangzhou, China). Full-length TRPM7 amplified by GenePharma Technology (Shanghai, China) was subcloned into pcDNA3.1 sites to generate the pcDNA3.1/TRPM7 plasmid. The abovementioned oligonucleotides and plasmids were individually transfected or cotransfected into cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.).

Normal human renal proximal tubule epithelial cell line HK-2 and RCC cell lines A498, 786-O, and Caki-2 were purchased from ATCC (American Type Culture Collection, Manassas, VA ,USA) and the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI 1640 medium (PAN Biotech, Aidenbach, Germany) supplemented with 10% foetal bovine serum (FBS) at 37 °C in a humidified atmospheric conditions of 5% CO2. As for cell transfection, miR-543 mimics and miR-543 inhibitors were constructed by Ribobio Company (Guangzhou, China). Transfections were performed using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer's protocol. Cells were harvested after 48 hours for further analyses.