Pgl3 baisc luciferase reporter vector

The specific DNA fragments containing the wild type or mutant PTTG3P fragment were sub-cloned downstream of the luciferase gene within the pGL3-Baisc luciferase reporter vector (GENECHEM Co., Ltd., Shanghai, China). Human A549 cells (1.0×10⁵) grown in a 24-well plate were co-transfected with 500 ng of either empty vector or E2F1 construct; 500 ng of firefly luciferase reporter comprising wild type or mutant PTTG3P using X-tremeGENE™ HP DNA (Roche, Shanghai, China) was used. Forty-eight hours after transfection, luciferase assay was performed by using the Dual-Luciferase Kit (Promega, USA). The relative firefly luciferase activities were normalized to those of Renilla luciferase. All samples were assayed in triplicate.

Downstream of the luciferase gene within the pGL3-Baisc luciferase reporter vector (GeneChem, China), the fragment of complementary DNA comprising the mutant or wild-type PVT1 fragment and 3’ untranslated region (UTR) of FAM193B was subcloned. 293T cell line of human (1.0×10⁵) seeded in a 24-well plate were implemented co-transfection with 150 ng of either miR-3127-5p and miR-328-3p or empty vector, firefly luciferase reporter of 50 ng containing mutant or wild-type PVT1 and 3’ UTR of FAM193B utilizing Lipofectamine 3000 (L3000-015, Invitrogen, USA). Luciferase analysis was assessed utilizing the DualLuciferase Kit (E1910, Promega, USA) 48 hours after transfection. To those of Renilla luciferase the relative activities of firefly luciferase were normalized. The transfection assay was echoed in triplicate.