Luciferase reporter plasmid

Lipofectamine 2000 (Invitrogen) was used to co-transfect MCF-7 and SKBR3 cells with a luciferase reporter plasmid (Promega, Madison, WI, USA) containing the wild-type or mutated 3′ UTR of the GPR30 gene downstream of the Renilla luciferase gene with a plasmid encoding hsa-WDR7-7 (XuanC Bio). After 48 h, luciferase activity was measured using a dual-luciferase assay system (Promega) and normalized to the activity of the Renilla luciferase internal control for transfection efficiency.

Human 624mel melanoma cells [30] (link) were transiently transfected at approximately 90% confluency at passage three in 6-well plates. Triplicates for each construct were performed for each experiment. 2 µg of luciferase reporter plasmid (Promega) and 50 ng of control Renilla plasmid (Promega) were transfected into each well of a 6-well plate utilizing 4 µl of Lipofectamine 2000 CD reagent (Invitrogen) in Opti-MEM medium (Gibco). The cells were lysed 24 h post-transfection and Firefly and Renilla luciferase activities were measured using the Dual Luciferase Reporter Assay System (Promega) with an Infinite M200 Luminometer (Tecan Munich GmbH, Kirchheim, Germany). Firefly values were divided by Renilla values to normalize for fluctuations in plated cells and transfection efficiency. Expression values of all test constructs were compared to the expression of the empty vector. Transfections were independently repeated four times per experiment. Values were transformed utilizing a transformation selector [31] (LN BASE e (X+1)) followed by a one way analysis of variance (Holm-Sidak method) (SigmaPlot v12).

To detect the regulatory relationship between tRF-5009A and mTOR mRNA, a dual-luciferase reporter assay was performed using SW1353 cells (a human chondrosarcoma cell line). The 3′-untranslated region (UTR) of mTOR mRNA with the tRF-5009A-binding site or its mutant construct was inserted into a luciferase reporter plasmid (Promega, USA). The luciferase reporter was cotransfected with the mTOR 3′-UTR fusion vector and the tRF-5009A mimic or corresponding NC into SW1353 cells. Cells were collected 48 h later and subjected to a dual-luciferase reporter assay (Promega, USA) using a Synergy H1 microplate reader (BioTek Instruments, USA) for the detection of firefly and Renilla luciferase activities.

To determine whether BRIP1 was the direct target of RP11-65M17.3, Lipofectamine 2000 (Invitrogen) was used to cotransfect HUVECs and HMEC-1 cells with a luciferase reporter plasmid (Promega) containing wild-type or mutated 3’-UTRs of BRIP1 with a plasmid encoding hsa-RP11-65M17.3 (XuanC Bio). Following transfection for 48 hours, the luciferase activity in each group was measured using a dual-luciferase assay system (Promega) and was normalized to the activity of Renilla luciferase.

The 3’-UTR of BACE1 and PRKACB containing the binding site of miR-200a-3p were cloned into the luciferase reporter plasmid (Promega; Madison, WI), and the binding site mutants were synthesized by a commercial company (Sangon Biotechnology, Shanghai, China). The WT or mutant luciferase plasmid together with the pRT-TK Renilla luciferase vector (Promega) were cotransfected with miR-200a-3p mimics or NCs into HEK293 cells. After 48 h, the corresponding vector’s luminescence was detected using the GloMax Multi luminometer (Promega) with a Dual-Luciferase Reporter Assay system. The Renilla luminescence was used to normalize the signal. All of the experiments were repeated four times independently.