Identification of an enhancer that increases miR-200b~200a~429 gene expression in breast cancer cells

The miR-200b~200a~429 gene cluster is a key regulator of EMT and cancer metastasis, however the transcription-based mechanisms controlling its expression during this process are not well understood. We have analyzed the miR-200b~200a~429 locus for epigenetic modifications in breast epithelial and mesenchymal cell lines using chromatin immunoprecipitation assays and DNA methylation analysis. We discovered a novel enhancer located approximately 5.1kb upstream of the miR-200b~200a~429 transcriptional start site. This region was associated with the active enhancer chromatin signature comprising H3K4me1, H3K27ac, RNA polymerase II and CpG dinucleotide hypomethylation. Luciferase reporter assays revealed the upstream enhancer stimulated the transcription of the miR-200b~200a~429 minimal promoter region approximately 27-fold in breast epithelial cells. Furthermore, we found that a region of the enhancer was transcribed, producing a short, GC-rich, mainly nuclear, non-polyadenylated RNA transcript designated miR-200b eRNA. Over-expression of miR-200b eRNA had little effect on miR-200b~200a~429 promoter activity and its production did not correlate with miR-200b~200a~429 gene expression. While additional investigations of miR-200b eRNA function will be necessary, it is possible that miR-200b eRNA may be involved in the regulation of miR-200b~200a~429 gene expression and silencing. Taken together, these findings reveal the presence of a novel enhancer, which contributes to miR-200b~200a~429 transcriptional regulation in epithelial cells.Joanne L. Attema, Andrew G. Bert, Yat-Yuen Lim, Natasha Kolesnikoff, David M. Lawrence, Katherine A. Pillman, Eric Smith, Paul A. Drew, Yeesim Khew-Goodall, Frances Shannon, Gregory J. Goodal

The miR-200 family is controlled by epigenetic-based mechanisms and mediates transition between non-stem and stem-like cell phenotypes.

MicroRNAs (miRNAs) are ~22 nucleotide (nt) single-stranded non-coding RNAs which are important regulators of gene expression in many biological processes including controlling cellular phenotype. The epithelial to mesenchymal transition (EMT) and the reverse process termed mesenchymal to epithelial transition (MET) are key programs that control the transition of cells between stem-like and non-stem phenotypes which are collectively termed epithelial plasticity. The miR-200 family is a key regulator of EMT however its role in controlling the transition between stem-like and non-stem phenotypes has not been well characterized. I utilized immortalized human mammary epithelial cells (HMLE) to investigate the function and regulation of the miR-200s during their conversion from a non-stem to a stem-like phenotype. HMLE cells were found to spontaneously convert from a non-stem to a stem-like phenotype. Isolation and comparison of the miR-200 expression between the spontaneously derived stem- like cells (sl-HMLE) and non-stem HMLE cells (nsl-HMLE) showed that the spontaneous conversion to a stem-like phenotype was accompanied by the loss of miR-200 expression. Likewise, miR-200 expression was also found to be down-regulated in prospective breast cancer stem cells (bCSCs) from metastatic pleural or ascites effusions and SUM159PT breast cancer cell line compared to non-CSC cells. This phenotypic change from a non-stem to a stem-like phenotype was directly controlled by the miR-200s as restoration of its expression partially converted the sl-HMLE cells to a non-stem phenotype with decrease stem-like properties and induction of an MET-like phenotype, although restoration of the miR-200 expression in SUM159PT prospective bCSCs did not have this effect. Next, using bioinformatic approaches and cell-based assays, I aimed to identify new miR-200 targets that are responsible for regulating the stem-like properties in both sl-HMLE cells and SUM159PT prospective bCSCs. Although the predicted genes (WNT5A, PKCα and PKCε) were not direct miR-200 targets, preliminary data suggest those genes may be involve in the survival or anoikis-resistance of stem-like cells and bCSCs. Investigation of the mechanism(s) controlling miR-200 expression revealed both DNA methylation and histone modifications were significantly altered in the stem-like and non-stem phenotypes. In particular, in the stem-like phenotype, the miR-200b/a/429 cluster was silenced primarily through polycomb group-mediated silencing whereas the miR-200c/141 cluster was repressed by DNA methylation. Furthermore, slight increase in EZH2 expression was observed in the stem-like phenotype and this might potentially contribute to the polycomb group-mediated silencing of the miR-200b/a/429 cluster. Lastly, preliminary co-immunoprecipitation results suggest that the targeting of polycomb group proteins to the miR-200b/a/429 promoter is not dependent on the ZEB1 transcription factor which is a repressor of the miR-200 transcription. Collectively, these results indicate that the miR-200 family plays a critical role in the transition between stem-like and non-stem phenotypes and that distinct epigenetic-based mechanisms regulate each miR-200 gene in this process. Therefore, combination of chemotherapy with therapies targeted against the miR-200 family members and epigenetic modifications would be beneficial towards treatment of breast cancer.Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 201

Transcriptomic analysis of the role of RasGEF1B circular RNA in the TLR4/LPS pathway

Abstract Circular RNAs (circRNAs) have recently emerged as a large class of novel non-coding RNA species. However, the detailed functional significance of the vast majority of them remains to be elucidated. Most functional characterization studies targeting circRNAs have been limited to resting cells, leaving their role in dynamic cellular responses to stimuli largely unexplored. In this study, we focus on the LPS-induced cytoplasmic circRNA, mcircRasGEF1B, and combine targeted mcircRasGEF1B depletion with high-throughput transcriptomic analysis to gain insight into its function during the cellular response to LPS stimulation. We show that knockdown of mcircRasGEF1B results in altered expression of a wide array of genes. Pathway analysis revealed an overall enrichment of genes involved in cell cycle progression, mitotic division, active metabolism, and of particular interest, NF-κB, LPS signaling pathways, and macrophage activation. These findings expand the set of functionally characterized circRNAs and support the regulatory role of mcircRasGEF1B in immune response during macrophage activation and protection against microbial infections

Inhibition of Euchromatic Histone Methyltransferase 1 and 2 Sensitizes Chronic Myeloid Leukemia Cells to Interferon Treatment

<div><p>Background</p><p>H3K9 methylation is one of the essential histone post-translational modifications for heterochromatin formation and transcriptional repression. Recently, several studies have demonstrated that H3K9 methylation negatively regulates the type I interferon response.</p><p>Results</p><p>We report the application of EHMT1 and EHMT2 specific chemical inhibitors to sensitize CML cell lines to interferon and imatinib treatments. Inhibition of EHMT1 and EHMT2 with BIX01294 enhances the cytotoxicity of IFNα2a in four CML cell lines, K562, KCL22, BV173 and KT1 cells. Chromatin immunoprecipitation assay shows that BIX01294 treatment enhances type I interferon response by reducing H3K9me2 at the promoters of interferon-stimulated genes. Additionally, BIX01294 treatment augments IFNα2a- and imatinib-mediated apoptosis in CML cell lines. Moreover, our data suggest that the expression level of EHMT1 and EHMT2 inversely correlates with the type I interferon responsiveness in CML cell lines.</p><p>Conclusions</p><p>Our study sheds light on the role of EHMT1 and EHMT2 as potential targets in improving the efficacy of standard treatments of CML.</p></div

BIX01294 slightly enhance IFNα2a-induced anti-proliferation in non-CML cells.

<p>Jurkat (<b>A</b>), HeLa (<b>B</b>) and HaCat (<b>C</b>) cells were cultured with various concentrations of BIX01294 and IFNα2a as indicated. After four days, cell proliferation was measured with a MTT assay. Results represent the mean ± SD in quadruplicate experiments.</p

BIX01294 enhances the expressions of ISGs in K562 cells.

<p>(<b>A</b>) K562 cells were incubated with 2.5 µM BIX01294 for 24 hours. The cells were then treated with various concentrations of IFNα2a as indicated. After two hours of IFNα2a stimulation, the expression of various ISGs was measured with RT-qPCR. Error bars represent the variation range of duplicate experiments. *: p<0.05, **: p<0.01. (<b>B</b>) K562 cells were incubated with 2.5 µM BIX01294 for 24 hours. The cells were then treated with IFNβ or IFNγ for two hours. The expression of <i>IFIT2</i> and <i>IFIT3</i> was measured with RT-qPCR. Error bars represent the variation range of duplicate experiments. *: p<0.05.</p

UNC06398 inhibits the proliferation of K562 cells and potentiates the expression of ISGs.

<p>(<b>A</b>) K562 cells were cultured with various concentrations of UNC0638 and IFNα2a as indicated. After four days, cell proliferation was measured with a MTT assay. Results represent the mean ± SD in quadruplicate experiments. (<b>B</b>) K562 cells were incubated with 5 µM UNC0638 for 24 hours followed with various concentrations of IFNα2a stimulation as indicated. After two hours of IFNα2a stimulation, the expression of various ISGs was measured with RT-qPCR. Error bars represent the variation range of duplicate experiments. *: p<0.05, **: p<0.01. (<b>C</b>) Whole cell extracts or total RNA were generated from K562 cells infected with control or lentiviruses carrying EHMT1- or EHMT2-specific shRNAs (left). EHMT1 or EHMT2 protein levels were analyzed by immunoblotting using indicated antibodies while mRNA levels were measured with RT-qPCR. Error bars represent the variation range of duplicate experiments. The same cells were stimulated with 1000 IU/ml IFNα2a for two hours (right). The expression of various ISGs was measured with RT-qPCR<b>.</b> Error bars represent the variation range of duplicate experiments. **: p<0.01. (<b>D</b>) K562 cells as in (<b>C</b>) were cultured with various concentrations of IFNα2a as indicated. After four days, cell proliferation was measured with a MTT assay. Results represent the mean ± SD in quadruplicate experiments.</p

BIX01294 inhibits the proliferation of CML cells.

<p>K562 (<b>A</b>), KCL22 (<b>B</b>), BV173 (<b>C</b>) and KT1 (<b>D</b>) cells were cultured with various concentrations of BIX01294 and IFNα2a as indicated. After four days, cell proliferation was measured with a MTT assay. Results represent the mean ± SD in quadruplicate experiments.</p

Expression level of EHMT1 inversely correlates with the sensitivity of CML cells to interferon.

<p>(<b>A</b>) KT1, K562, KCL22 and BV173 cells were treated with or without 1000 IU/ml IFNα2a for 2 hours, the expression of <i>IFIT2</i> and <i>IFIT3</i> was measured with RT-qPCR. Error bars represent the variation range of duplicate experiments. *: p<0.05, **: p<0.01. (<b>B</b>) KT1, K562, KCL22 and BV173 cells were incubated with or without 2.5 µM BIX01294 for 24 hours. Cells were then infected with VSV-GFP at a MOI of 0.5 for 24 hours. GFP positive cells were sorted by FACS. Results represent the mean ± SD in triplicate experiments (<b>C)</b> Whole cell extracts were prepared from K562, KT1, BV173 and KCL22 cells, and examined by immunoblotting using the indicated antibodies. (<b>D</b>) The relative mRNA levels of EHMT1 and EHMT2 were measured with RT-qPCR. Results represent the mean ± SD in quadruplicate experiments. *: p<0.05. (<b>E</b>) Empty vector or FLAG-mEHMT1-HA-mEHMT2 KT1 cells were treated with or without IFNα2a (1000 IU/ml) for two hours, the expression of <i>IFIT2</i> and <i>IFIT3</i> was measured with RT-qPCR<b>.</b> Error bars represent the variation range of duplicate experiments. **: p<0.01.</p