BART miRNAs alter gene expression in THP-1 cells. (A) BART miRNAs were overexpressed in THP-1 cells with the Tet-Off system. (B) BART miRNA expression was regulated by doxycycline. After 7 to 9 days of culture, the number of cells was counted. Cell proliferation is expressed as the fold-change relative to the cell number at day 0. *P < .05, using Student t test, compared with control values of each doxycycline concentration. (C) Total RNA was collected from BART/THP-1 cells at the indicated time and doxycycline concentration. TNF-α mRNA expression level was measured by qPCR. **P < .01, using Student t test, compared with day 0. (D) Microarray data revealed that about 400 genes were upregulated and 100 genes were downregulated by more than 2-fold in BART/THP-1 cells relative to both control cells treated with doxycycline (control) and cells treated with empty vector (Empty). (E) Microarray results were validated by RT-qPCR. Expression of ARG1 and RILP were upregulated by induction of BART miRNAs. Expression of CIITA was downregulated by induction of BART miRNAs. Expression levels were normalized to GAPDH. Averages from 3 independent experiments were shown. *P < .05, Student t test. (F) 3′-UTR luciferase assay was performed with HEK293T cells (left). Either the empty vector or BART miRNA overexpression vector was cotransfected along with the psiCHECK^TM-2 vector, a luciferase reporter vector. Ctrl, psiCHECK^TM-2 vector; WT, inserted wild-type 3′-UTR sequence of target genes; mut., including mutated target sites. *P < .05, Student’s t-test. Position of the predicted target sequences (miRANDA) of BART miRNAs in the 3′-UTR of human MEF2C mRNAs (right). (G) Downregulation of MEF2C by siRNA treatment enhanced LPS-stimulated expression of IL-10. Expression level was normalized to GAPDH. Three siRNAs were mixed to minimize off-target effects. An average of 3 independent experiments was shown. *P < .05, Student t test.