Luciferase reporter vector

TargetScan program was employed for prediction of target gene of miR-14. The 3′-UTR of candidate gene was cloned into luciferase reporter vector according to the provider’s instruction (Promega). Both M14 and MV3 cells were seeded into six-well plate the day before transfection. Co-transfection of luciferase reporter plasmid and miR-14 was performed with Lipofectamine 2000 (Invitrogen). Relative luciferase activity was measured by micro-plate reader (Molecular Devices, Sunnyvale, CA).

The potential binding site of miR-4735-3p in SCL40A1 3′-UTR was obtained from TargetScan (http://www.targetscan.org/vert_71/), and then, the predicted sequence was cloned into the luciferase reporter vector (Promega, Madison, WI, USA). HEK293T cells were cotransfected with the miR-4735-3p mimic and luciferase reporter plasmid, and relative luciferase reporter activity was determined at 48 h using Dual Luciferase Reporter Assay System (Promega) [41 (link), 42 (link)].

The synthesized promoter regions (−1000 bp and −500 bp) of the miR-222 gene were subcloned into the luciferase reporter vector (Promega, USA). Luciferase reporter constructs were packed with an adenoviral system and then cotransfected into NRCMs with a control plasmid, followed by the indicated stimulation: Ad-HMGA1 transfection or siHMGA1 for 48 h. Then, the cells were harvested and lysed, and the luciferase activity was determined with the Dual-Luciferase Reporter Assay Kit (Promega, USA) according to the manufacturer’s instructions. The PGL3 basic vector was used as a negative control.

A549 and H1299 cells were co-transfected with RUNX3-OE plasmid or empty vector control and Suv39H1-WT or -MUT. Lipofectamine 2000 (Invitrogen) was used for transfection. After 48 h of transfection at 37 °C with the luciferase reporter vector (Promega), a Dual Luciferase Reporter Assay kit (Promega) was used to evaluate the relative luciferase activities. The Renilla luciferase reporter was used as internal control. The activities of firefly luciferase and Renilla luciferase were quantified by using the dual luciferase reporter assay system (Promega).

Using TargetScan 7.2 software, DTL was predicted to be a target gene of miR-96-5p. The full-length 3′ untranslated region (UTR) of DTL was amplified by PCR and cloned at the SacI and XhoI sites into a luciferase reporter vector (Promega, Madison, WI, USA). The mutant or wild-type construct of DTL 3′UTR was constructed by RiboBio (RiboBio Co., Guangzhou, China). HEK293T cells in 96-well plates were co-transfected with a reporter construct (pmiR-null Report plasmid, pmiR-DTL 3′UTR-wt, pmiR-DTL 3′UTR-mut) and miR-96-5p mimics or miR-negative control (NC) using Lipofectamine 3000 reagent (Invitrogen, Waltham, MA, USA). A dual-luciferase reporter assay system (Promega, Madison, WI, USA) was used to measure the levels of luciferase activity after 48 h. The results were normalized by dividing the firefly luciferase activity by the Renilla luciferase activity in accordance with the manufacturer’s instructions.