Pgl3 luciferase reporter vector

StarBase database was used to predict the interactions of circ-MEMO1-miRNAs and miR-101-3p-mRNAs. Dual-luciferase reporter assay was used to test the target relationship between miR-101-3p and circ-MEMO1 or KRAS. The partial sequence in circ-MEMO1 or the 3′ untranslated region (3′ UTR) of KRAS messenger RNA (mRNA), containing the binding sites with miR-101-3p, was directly amplified and inserted into pGL3 luciferase reporter vector (Promega, Madison, WI, United States), termed as circ-MEMO1-wt or KRAS 3′ UTR wt. Meanwhile, the corresponding mutant sequence in circ-MEMO1 or KRAS was also amplified and inserted into pGL3 luciferase reporter vector (Promega) to generate circ-MEMO1-mut or KRAS 3′ UTR mut using Site-directed gene mutagenesis kit (Takara, Dalian, China). After co-transfecting these reporter constructed plasmids (50 ng) and miR-NC or miR-101-3p (20 nM) into NSCLC cells for 48 h, the luciferase activity was detected with the dual-luciferase reporter assay system (Promega) using the luminometer (Plate Chameleon V, Hidex, Finland). Firefly luciferase activity was normalized to Renilla luciferase intensity. The experiment was repeated three times.

The targets of circ_0007841 and miR-338-3p were predicted by circinteractome and targetscan software, respectively.
The wild-type partial sequence in circ_0007841 that predicted to bind to miR-338-3p, along with the mutant-type sequence with miR-338-3p in circ_0007841 that was synthesized through using Site-directed gene mutagenesis kit (Takara, Dalian, China), was amplified and cloned into pGL3 luciferase reporter vector (Promega, Madison, WI, USA), termed as circ_0007841 WT or circ_0007841 MUT. MM cells were co-transfected with 10 nM miR-NC or miR-338-3p and 40 ng circ_0007841 WT or circ_0007841 MUT. After 48-h transfection, MM cells were harvested and the luciferase activity was detected with the dual-luciferase reporter assay system kit (Promega) using the luminometer (Plate Chameleon V, Hidex, Finland) according to the manufacturer’s instructions. Firefly luciferase activity in each group was normalized to Renilla fluorescence intensity.
The wild-type fragment of BRD4 3′ untranslated region (3′UTR) that predicted to bind to miR-338-3p and the mutant type fragment of BRD4 3′UTR were also amplified and inserted into pGL3 luciferase reporter vector (Promega) to generate BRD4 3′UTR WT and BRD4 3′UTR MUT. Co-transfection of MM cells with BRD4 3′UTR WT or BRD4 3′UTR MUT and miR-NC or miR-338-3p was conducted following the similar procedure.

Human SIRT1 3′ UTR contained with seed region of miR-29b was cloned into pGL3 luciferase reporter vector (Promega, USA) downstream of the luciferase gene. The primer sequences are as follows: forward: 5′-GGGGTACCATAATTTTTAACTTCATTAT-3′, reverse: 5′-GAAGATCTTATTACAAGTTACATCAT CAT-3′. For luciferase reporter assay, 50 pmol/ml miR-29b, 2 μg/ml recombinant pGL3 luciferase reporter vector and 100 ng/ml renilla luciferase pRL-TK vector (Promega) were co-transfected into SW480, OR-SW480 and OR’-SW480 cells. 24 h after transfection, cells were collected and lysed. Activities of pGL3-firefly luciferase and pRL-TK-renilla luciferase were analyzed by using dual-luciferase reporter assay system (Promega) according to the manufacturer’s protocol. Firefly luciferase activities were normalized to renilla luciferase activities