Signal transducer and activator of transcription (STAT)-3 regulates microRNA gene expression in chronic lymphocytic leukemia cells

Abstract

Backgrounds: Approximately 1,000 microRNAs (miRs) are present in the human genome; however, little is known about the regulation of miR transcription. Because miR levels are deregulated in chronic lymphocytic leukemia (CLL) and signal transducer and activator of transcription (STAT)-3 is constitutively activated in CLL, we sought to determine whether STAT3 affects the transcription of miR genes in CLL cells.

Methods: We used publically available data from the ENCODE project to identify putative STAT3 binding sites in the promoters of miR genes. Then we transfected CLL cells with STAT3-shRNA or with an empty vector, and to determine which miRs are differentially expressed, we used a miR microarray approach followed by validation of the microarray results for 6 miRs using quantitative real-time polymerase chain reaction (qRT-PCR).

Results: We identified putative STAT3 binding sites in 160 promoter regions of 200 miRs, including miR-21, miR-29, and miR-155, whose levels have been reported to be upregulated in CLL. Levels of 72 miRs were downregulated (n = 63) or upregulated (n = 9). qRT-PCR confirmed the array data in 5 of 6 miRs.

Conclusions: The presence of activated STAT3 has a profound effect on miR expression in CLL cells.

Figure 1

Transfection with STAT3-shRNA alters miR gene expression in CLL cells. A. Left panel: Quantitative RT-PCR shows an 8 fold reduction in STAT3 mRNA levels in CLL cells transfected with STAT3-shRNA compared to control CLL cells transfected with an empty vector. The means ± S.D. of 3 experiments are depicted. Right panel: Western immunobloting shows a decrease in STAT3 protein levels of CLL cells transfected with STAT3-shRNA compared to untreated cells or cells transfected with an empty vector. B. MiR array heat map depicts 23 miRs whose expression differed significantly between CLL cells transfected with STAT3-shRNA and CLL cells transfected with empty vector. Red, high levels; blue, low levels. C. Panels a through f show the results of RT-PCR analysis of CLL cells transfected with STAT3-shRNA. The plots show relative expression as means ± SD of 3 replicates for each miR tested. Shown are relative expression of miRs in the unsorted STAT3-shRNA transfected CLL cells (transfection efficiency: 30%; middle, gray bars), sorted STAT3-shRNA-transfected CLL cells (black bars), and empty vector-transfected cells (light gray bars labeled “control”).