Luciferase Reporter Assay

We used RNAhybrid and miRanda to predict miR-222 target genes and chose NASP, LHX8, BCL2L11 and EGFR for a luciferase reporter assay. The wild-type gene fragments of the 3’UTR region were amplified by PCR with an upstream primer within the SacI site and a reverse primer within the XbaI site using PrimeSTAR polymerase (Takara, China, Japan), and purified with a TIANgel Midi Purification Kit (Tiangen, China). The products were digested by the restriction endonucleases SacI and XbaI (TransGen, China), and the inserts were linked to the pmirGLO double luciferase reporter vector with a DNA ligation kit (Takara, China). The constructs were validated by sequencing. Mutant vectors were created from the wild-type vectors by using a Fast Mutagenesis System (TransGen, China). We designed mutation primers (Supplementary Table 3) to change the seed sequences (the miRNA-gene binding sites). The resulting reporter vectors are termed NASP-WT, LHX8-WT, BCL2L11-WT and BCL2L11-MUT, EGFR-WT and EGFR-MUT.

The constructed plasmids were cotransfected with the miR-222 mimic or inhibitor into 293T cells using Lipofectamine 2000 (Invitrogen, United States). Forty-eight hours after transfection, luciferase activity was detected by an enzyme labeling instrument (Tecan, Switzerland) according to the specifications of the Dual Luciferase Reporter Assay System (Promega, United States).