Effect of deregulated miRNAs on expression of their target genes. (A) miR-101 represses STMN1. Top: Reintroduction of miR-101 into NKYS cells reduced mRNA levels of STMN1. mRNA levels of STMN1 were determined by quantitative RT-PCR analysis. Cells were transduced by lentivirus produced by miRNA precursor expression vectors or a control vector. Cells were harvested 4 days after transduction. Mature miR-101 transcript was determined by TaqMan miRNA assay. Bottom: 3′-UTR luciferase reporter assay of STMN1. The 293T cells were cotransfected with a reporter construct with or without 3′-UTR of STMN1 that contain 2 potential miR-101 binding sites and an miRNA expression vector with or without hsa-miR-101 precursor sequence. Reporter constructs with point mutations to the seed sequence of either miR-101 target site were similarly cotransfected. Luciferase activity was determined 48 hours after transfection. (B) miR-30b represses PRDM1. Top: Reintroduction of miR-30b into NK-YS cells reduced mRNA levels of PRDM1. Cells were transiently transfected by miRNA-30b mimics. Expression of PRDM1 at 48 hours after transfection was determined by quantitative RT-PCR analysis. Bottom: 3′-UTR luciferase reporter assay of PRDM1. The 293T cells were cotransfected with a reporter construct with or without the whole 3′-UTR of PRDM1 cloned to the distal end of the firefly luciferase gene and an miRNA expression vector with or without the hsa-miR-30b precursor sequence. Luciferase activity was determined 48 hours after transfection. (C) Re-expression of miR-101, miR-26a, or miR-26b in NK-YS cells reduced expression of BCL-2. Cells were transduced by lentivirus produced by miRNA precursor expression vectors or a control vector and were harvested 4 days after transduction for quantitative RT-PCR analysis.