Pgl4 reporter plasmid

Bioinformatics analysis was performed to predict the interaction between miR-455-3p and circCSNK1G1 or MYO6 and analyze the binding sites between them using the online database starbase (http://starbase.sysu.edu.cn/). According to the predicted wild-type sequence of binding sites, the sequence of circCSNK1G1 or MYO6 mutated at the binding sites was designed. Then, the wild-type and mutant-type sequences of circCSNK1G1 or MYO6 3ʹ-UTR were amplified and cloned into PGL4 reporter plasmid (Promega, Madison, WI, USA), namely circCSNK1G1 WT, circCSNK1G1 MUT, MYO6 3ʹ-UTR-WT or MYO6 3ʹ-UTR-MUT, respectively. Subsequently, the alone fusion plasmid and miR-455-3p or miR-NC were cotransfected into HCT116 and SW620 cells. After 48 h, the luciferase activity was detected using the Dual-Luciferase Assay System (Promega).

According to the wild-type sequence of circUSP36 or ROCK2 3′UTR containing miR-20a-5p binding sites, the mutant sequence of circUSP36 or ROCK2 3′UTR (binding sites mutation) was designed. These sequences (wild-type and mutant-type) were amplified and separately inserted into pGL4 reporter plasmid (Promega Corporation) to generate luciferase reporter plasmids, which were named as WT-circUSP36, MUT-circUSP36, WT-ROCK2 and MUT-ROCK2, respectively. WT-circUSP36 (2 µg), MUT-circUSP36 (2 µg), WT-ROCK2 (2 µg) or MUT-ROCK2 (2 µg) together with miR-20a-5p mimic (40 nM) or miR-NC mimic (40 nM) were co-transfected into the HUVECs using Lipofectamine 3000 kit (Invitrogen; Thermo Fisher Scientific, Inc.). Transfected cells were then incubated for 48 h at 37°C, and the luciferase activities in these cells were investigated using the dual-luciferase reporter system (Promega Corporation) according to the manufacturer's protocol. Relative luciferase activity was expressed as normalization of Renilla luciferase activity to Firefly luciferase activity.