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BET inhibition modulates miRNAs in DLBCL
to a perturbation of its downregulation by PRMT5. To assess this, we performed qRT-PCR analysis of PRMT5 in DLBCL cells treated with OTX015 for 4, 24 and 48 h. PRMT5 mRNA was markedly downregulated at 4 and 24 h. At 48 h PRMT5 levels were similar in DMSO- and OTX015-treated cells for all four cell lines (Figure 2B). At the protein level (Figure 2C), moderate downregulation of PRMT5 was evident at 24 h in DOHH-2 cells treated with OTX015. In SU-DHL-2 cells, PRMT5 was moderately downregulated at 24 h and was negligible at 48 h. These results indicate that OTX015 could downregulate the lev- els of expression of both PRMT5 RNA and protein in DLBCL cells. Hence upregulation of miR-96-5p following OTX015 treatment was associated with downregulation of PRMT5 protein, particularly in SU-DHL-2 cells.
A
BRD4 binds to the 5’ regulatory region of PRMT5
in diffuse large B-cell lymphoma cells and treatment with a BET inhibitor reduces BRD4 binding
As OTX015 mediates transcriptional repression by dis- placing BRD4 from chromatin,15,21 we hypothesized that PRMT5 was transcriptionally regulated by BRD4. To assess this, we re-analyzed the two public ChIP sequenc- ing datasets of DLBCL cells treated with the BET inhibitor JQ1,22,23 which has a similar mechanism of action to OTX015 and exhibits an overlapping profile of targeted genes.15,22 This revealed that BRD4 bound to the 5’ region of PRMT5 and that this binding was reduced following treatment with the BET inhibitor (Figure 3A). In agreement with the public ChIP sequencing data of JQ1-treated DLBCL cells, when we performed ChIP-
B
Figure 1. BRD4 binds to the regulatory regions of microRNAs. (A) The genomic regions within ± 1 kb of miRNA promoters that are bound by BRD4 obtained using ngs.plot. Lines represent the average expression profiles of “expressed” (red line) and “not expressed” (green line) miRNA. (B) Analysis of publicly available chromatin immunoprecipitation sequencing data of diffuse large B-cell lymphoma (DLBCL) cells showed that BET inhibitor treatment reduces BRD4 binding at the 5’ regulatory regions of the miR-17-92 cluster, which contains miR-92a-1-5p, and the miR-155 host gene (left panel). An activated B-cell (ABC)-DLBCL cell line (SU-DHL-2) and a ger- minal center B-cell (GCB)-DLBCL cell line (DOHH-2) were treated with OTX015 for 4 and 24 h before TaqMan quantitative reverse transcription polymerase chain reaction analysis of miR-92a-1-5p and miR- 155-5p expression (right panel). miR-155- 5p expression is only shown for SU-DHL-2 as it is an ABC-DLBCL specific oncomiR. Expression of RNU6B was used for normal- ization. For each timepoint, the mean fold- change relative to the dimethyl sulfoxide (DMSO) control is shown. Charts show the mean of at least three independent experi- ments. *P<0.05; **P<0.01. Error bars denote the standard error.
haematologica | 2018; 103(12)
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