Validating miR-155 Regulation of SOCS1

The biological prediction website microRNA.org was employed to analyze the target genes of miR-155, and dual-luciferase reporter gene assay was employed to verify that the candidate SOCS1 was a direct target gene of miR-155. The SOCS1-3’-untranslated region (UTR) sequence and the mutated SOCS1-3’-UTR sequence were synthesized. The two synthesized target gene fragments were cloned into the pmir GLO dual-luciferase reporter gene vector to construct the SOCS1-3’-UTR dual-luciferase reporter wild-type gene vector (pmir GLO-SOCS1) and its mutant vector (pmir GLO-mut-SOCS1). The recombinant vector was investigated by polymerase chain reaction (PCR), electrophoresis, and gene sequencing to prove that it had been successfully constructed. HEK-293T cells (Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China) were respectively co-transfected with the two recombinant vectors and miR-155 mimic or miR-155 NC by using the transfection reagent Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After 48 hours of transfection, cells were lysed. The luciferase activity was detected on a Luminometer TD-20/20 detector (E5311, Promega, Madison, WI, USA) using a Dual-Luciferase^® Reporter Assay System kit (Promega, Madison, WI, USA) (Song et al., 2017 (link)).