Signal Transducer and Activator Of Transcription 3 Activates and Deregulates Micro RNA Expression In Chronic Lymphocytic Leukemia Cells

MicroRNA (miR) deregulation is a hallmark of chronic lymphocytic leukemia (CLL). However, little is known about the mechanisms of miR gene transcription. In CLL, signal transducer and activator of transcription 3 (STAT3) is constitutively phosphorylated on serine 727 residues, and phosphoserine STAT3 activates protein-coding genes known to be induced by phosphotyrosine STAT3. Therefore, we sought to determine whether phosphoserine STAT3 also activates miR genes. Because an analysis of publically available data, revealed that STAT3 binds to the promoter of nearly 25% of all human miR genes, we transfected unstimulated CLL cells with STAT3 small hairpin (sh)-RNA. Using an RNA microarray, we found that STAT3-shRNA downregulated the levels of 63 miR genes and upregulated the levels of 9 miR genes. Using qRT-PCR, we confirmed the microarray results in 5 of the 6 differentially expressed miR genes. Together, these data suggested that STAT3 affects miR gene levels. An analysis of chromatin immunoprecipitation followed by sequencing (ChIP-seq) data revealed STAT3-miR promoter binding sites in only 60% of the STAT3-regulated miR genes. This suggested to us that mechanisms unrelated to miR promoter binding are involved in the STAT3-mediated regulation of miR expression. Indeed, computer simulation of complementary binding of STAT3-targeted microRNA genes to STAT3 mRNA provided indirect evidence that STAT3 mRNA acts as a “sponge”, soaking up miRs and altering their level and function. Several miRs that are downregulated by STAT3-shRNA, including miR-15, miR-16, miR-21, and miR-155, play crucial roles in CLL pathobiology. In particular, miR-155 is involved in tumorigenesis, and its overexpression induces lymphoma in mice; also, CLL has a high expression level of miR-155. Therefore, we sought to confirm that STAT3 directly regulates the transcription of miR-155 in CLL cells. Electrophoretic mobility shift assay analysis revealed that STAT3 bound to the miR-155 promoter in CLL cells, and ChIP analysis revealed that STAT3 bound the 700–709 bp but not the 615–624 bp in the miR-155 promoter. We then co-transfected MM1 cells with truncated miR-155 promoter constructs plus STAT3 small interfering (si)-RNA and found that the STAT3-siRNA reduced the luciferase activity of the construct harboring the 700–709 bp miR-155 promoter region. Taken together, our data suggest that STAT3 directly and indirectly deregulates miR gene levels in CLL.