Site directed gene mutagenesis kit

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.

The Xist fragment containing the putative miR-32-5p binding site was cloned into the pmirGlo vector (Promega, United States) and named WT Xist. Mutation of the putative miR-32-5p target sequence in the 3-UTR of Xist was generated using a site-directed gene mutagenesis kit (Takara) and named MUT Xist. HEK-293T cells were co-transfected separately with the two constructs or empty vector and miR-32-5p mimics or inhibitors. For miR-32-5p and Notch-1 interaction, the putative and mutated miR-32-5p target binding sequences in Notch-1 were synthesized and cells were co-transfected with the WT or MUT Notch-1 reporter gene plasmid or pmirGlo plasmids using Lipofectamine^® 2000 (Invitrogen). At 48 h post-transfection, cells were harvested and the firefly luciferase activity was measured using a dual-luciferase reporter assay system (Promega) and normalized to Renilla luciferase activity (Jin et al., 2018 (link); Hu et al., 2019 (link)).

The bioinformatics software StarBase (https://starbase.sysu.edu.cn/) was used to predict the potential-binding sites of PEG10-miRNA interactions and miR-449a-miRNA interactions. The target interactions between miR-449a and PEG10 or RPS2 were confirmed by Dual-luciferase reporter assay. The predictive wild type (WT) of binding sequences with miR-449a in PEG10 and RPS2 were amplified and cloned into pmirGLO vector (Promega), called as PEG10-WT and RPS2-WT. Simultaneously, the mutant type (MUT) of binding sequences in PEG10 and RPS2 were also synthesized by site-directed gene mutagenesis kit (Takara, Japan) and inserted into pmirGLO vector (Promega), named as PEG10-MUT and RPS2-WT. SK-N-BE (2) and SH-SY5Y cells were inoculated into 24-well plates for 24 h, then transfected with 50 ng of reporter plasmids along with 20 nM of miR-449a or mimic NC with Lipofectamine 3000 (Invitrogen). After transfection for 48 h, cells were lysed, luciferase activities were assessed by a Dual-Luciferase Reporter Assay Kit (Promega) in light of the instructions of manufacturer and normalized with Renilla luciferase as control [28 (link)].

The 3′-UTR of human EGFR, including the complementary binding site of miR-539, was cloned downstream of the firefly cassette of the psiCHECK-2 dual-luciferase reporter plasmid (Promega, WI, USA) to construct the psiCHECK-2-EGFR wild-type (WT) luciferase reporter plasmid. The complementary binding site was then mutated using a site-directed gene mutagenesis kit (TaKaRa, Japan). The mutated EGFR 3′-UTR was also inserted into the psiCHECK-2 plasmid to generate the psiCHECK-2-EGFR mutant (MUT) luciferase reporter plasmid. MDA-MB-231 and MCF7 cells were co-transfected with psiCHECK-2-EGFR (WT) or (MUT) and miR-539 mimics or the mimic control using Lipofectamine 2000 according to the manufacturer’s protocols. Forty-eight hours after transfection, a dual-luciferase reporter assay system (Promega, WI, USA) was used to detected the firefly and Renilla luciferase activities. The firefly luciferase activities were normalized to the Renilla luciferase activities.