Lentiviral Transduction and 3'UTR Luciferase Assay

Lentivirus construction and infection were performed as formerly introduced (24 (link)). In brief, after digestion with EcoR I and Xba I (Takara, Dalian, China), pre-miR-486-5p sequence was cloned into pLenti vector (Invitrogen, Carlsbad, CA, USA) (named pLenti-miR-486). Viral particles containing pLenti or pLenti-miR-486 were collected to infect A549 and H1299 cells and further sorted with flow cytometry.
The wild-type (WT) RSK and p70S6K 3’UTR fragments and their corresponding mutant (Mut) fragments were cloned into pGL3-basic (pGL3, Promega, Madison, WI, USA) luciferase reporter vector to confirm their direct binding with miR-486-5p. These pGL3-derived vectors and miR-486-5p mimics were then cotransfected into 293T cells, and the Dual-Luciferase Reporter Assay (Promega, Madison, WI, USA) was applied for luciferase activity measurement as previously described (25 (link)).
The sequences of RSK and p70S6K were constructed into the pcDNA3.1 plasmid. siRNAs for RSK (siRSK #1-3) and p70S6K (sip70S6K #1-3) were synthesized by RIBOBIO (Guangzhou, China). All siRNA sequences were listed in Table S2. Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) was used for transient transfection. All the constructed vectors were sequenced (Sangon Biotech, Shanghai, China) and the primers are listed in Table S3.