Luciferase reporter assay was used to detect the direct binding between circRNA-100290 and miRNAs. pmiR-RB-Report vector (Saierbio, Tianjin, China) containing renilla luciferase gene (hRluc) and firefly luciferase gene (hLuc+) was applied in this experiment. The 3′ UTR sequence of circRNA_100290 was cloned downstream of hRluc cassette. Mutations were performed in the binding sites. The miRNA mimics were obtained from GenePharma (Shanghai, China). hLuc+ cassette was used as internal control. Each miRNA or negative control oligonucleotide was co-transfected with pmiR-RB-Report vector with or without the 3′UTR sequence of circRNA_100290. Finally, the relative light units (RLU) of hRluc and hLuc+ were determined by Veritas 9100-002 (Turner BioSystems, Sunnyvale, CA, USA), and the hRluc values were normalized to the corresponding hLuc+ values.
An EGFP/RFP reporter assay was used to confirm that CDK6 is a direct target of the miR-29 family. miR-29b mimics or negative control oligonucleotide was co-transfected with pcDNA3 reporter vector containing the wild-type 3′UTR of CDK6 or a mutant version of the 3′UTR of CDK6. The expression levels of EGFP were normalized by the RFP values. The expression values of EGFP and RFP were determined by an F-4500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan).
An EGFP/RFP reporter assay was used to confirm that CDK6 is a direct target of the miR-29 family. miR-29b mimics or negative control oligonucleotide was co-transfected with pcDNA3 reporter vector containing the wild-type 3′UTR of CDK6 or a mutant version of the 3′UTR of CDK6. The expression levels of EGFP were normalized by the RFP values. The expression values of EGFP and RFP were determined by an F-4500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan).