K801 200

The synthesized MDM2 3′ untranslated region (UTR) gene fragment MDM2-WT and the MDM2-MUT mutated at the binding site were constructed into a pMIR-reporter plasmid (Beijing Huayueyang Biotechnology, Beijing, China). Luciferase reporter plasmids were co-transfected with miR-9-3p into HEK293T cells (Shanghai Beinuo Biotechnology, Shanghai, China). Then 48 h subsequent to transfection, cells were lysed, and detected using a luciferase detection kit (K801-200; Biovision, Mountain View, CA, USA).

The biological prediction website microRNA.org was used to analyze target genes of miR-132. We verified whether NRF2 was a direct target gene of miR-132 using a dual-luciferase reporter gene assay. Artificially synthesized NRF2 3′ untranslated region (3′UTR) gene fragments were introduced into the reporter plasmid pMIR-reporter (Beijing Huayueyang Biotechnology Co., Ltd., Beijing, China) using the endonuclease sites SpeI and Hind III. The complementary sequence mutation sites of the seed sequence were designed based on the wild-type (WT) NRF2. T4 DNA ligase was used to insert the target fragments into the pMIR-reporter plasmid after digestion with restriction enzymes. The correctly sequenced luciferase reporter plasmids NRF2 WT and NRF2-mutant (MUT) were cotransfected into T24 cells with miR-132 mimic. In addition, oe-KLF8, oe-NEDD4 or empty plasmids were cotransfected with the KLF8-mediated CyclinD1 promoter sequence into T24 cells. Cells were harvested 48 h later to extract the proteins. The luciferase detection kit (K801-200, Biovision) of GloMax20/20 Luminometer (Promega, Madison, WI, USA) was employed to assess luciferase activity.

The wt and mut reporter plasmids of HOTAIR (wt-HOTAIR; mut-HOTAIR) and NPTX2 (wt-NPTX2 and mut-NPTX2) were designed by Shanghai GenePharma Co., Ltd. (Shanghai, China). NC mimic or miR-221-3p mimic was co-transfected with wt-HOTAIR, mut-HOTAIR, wt-NPTX2 or mut-NPTX2 into the MN9D cells. After 24 h, the cells were treated with 100 μmol/L MPP⁺ (Sigma-Aldrich Chemical Company, St Louis, MO, USA) or PBS for 24 h and then lysed. The luciferase activity of the target reporter gene was analyzed according to the instructions of the luciferase detection kit (K801-200, BioVision, Milpitas, CA, USA).

The KLF2 3′ untranslated region (3′UTR) fragment, containing binding sites to miR‐144, was inserted into the pGL3 plasmid. By using the point mutation method, the KLF2‐3′UTR‐mutant (MUT) fragment containing mutated binding sites was constructed and inserted into the pGL3 plasmid. Through liposome transfection, the correctly sequenced pGL3‐KLF2‐wild‐type (WT) and pGL3‐KLF2‐MUT recombinant vector were cotransfected into HEK293T cells with either miR‐144 mimic or NC mimic. The cells were collected and lysed after being transfected for 48 hours, and the relative light unit of each sample was detected using a luciferase detection kit (K801‐200, Biovision) at dual‐luciferase reporter gene analysis system (Promega). The relative luminescence activity was determined as the ratio of the firefly luciferase relative light unit (RLU) to the Renilla luciferase RLU (internal reference).