Unknown,Transcriptomics,Genomics,Proteomics

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MiR-18a modulates gene transcription in breast cancer cells treated with Cobalt(II) chloride

ABSTRACT: Using the orthotopic breast cancer xenograft model of basal-like breast cancer MDA-MB-231 line, we have found that the expression of miRNAs encoded by MIR17HG was significantly decreased in cells isolated from spontaneous lung metastases compared to cells from primary tumors grown in orthotopic sites. We investigated the role of a MIR17HG family member, miR-18a, in primary tumor growth and pulmonary metastasis from the orthotopic site. We demonstrated that enforced expression of exogenous miR-18a, significantly limited continuous growth of primary tumors in mammary gland fat pads and reduced spontaneous lung metastasis. Further investigation on the mechanism of miR-18a action led to the finding that the expression of HIF1A, a key regulator of tumor metastasis, was regulated by miR-18a. Enforced miR-18a expression significantly decreased HIF1A expression at both mRNA and protein levels, resulting in altered transcriptional response and decreased survival of cells in response to Cobalt(II) chloride (CoCl2), a hypoxia-mimicking agent. Conversely, miR-18a knockdown significantly increased HIF1A expression levels and enhanced cell survival in response to CoCl2. Analysis of expression data of human breast tumor tissues showed that miR-18a expression is inversely correlated with HIF1A expression in basal-like breast tumors, supporting a role of miR-18a in restricting HIF1A expression in this subtype of breast cancer. In addition, we demonstrated that hypoxia inhibits miR-18a expression, likely through MYC inactivation. Furthermore, gene expression and functional analysis revealed that miR-18a also plays a role in regulating cell adhesion, migration and invasion. Taken together, this study provides evidence for a novel role of miR-18a to inhibit breast cancer metastasis. Our results suggest that miR-18a downregulation might provide tumor cells survival/growth advantage under hypoxic pressure in basal-like breast cancer. A lung metastatic subline, designated as MB231RN-LM, was derived from MDA-MB-231 breast cancer cells through in vivo selection. The MB231RN-LM cells were stably transfected with has-miR-18a or control vector and treated with 200uM Cobalt(II) chloride (CoCl2, a hypoxia-mimicking agent) for 4 hr. A total of 8 samples were subjected to microarray analysis, with two biological repeats for each experiment condition.

ORGANISM(S): Homo sapiens

SUBMITTER: Meiyun Fan

PROVIDER: E-GEOD-45362 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose The analysis of breast cancer residual tumors after neoadjuvant chemotherapy (nCT) may be useful for identifying new biomarkers. MicroRNAs are known to be involved in oncogenic pathways and treatment resistance of breast cancer. Our aim was to determine the role of miR-18a, a member of the miR-17-92a cluster, in breast cancer behavior and outcome after nCT. Methods Pre- and post-nCT tumor miR-18a expression was retrospectively assessed by qRT-PCR in 121 patients treated with nCT and was correlated with survival outcomes and with clinical and pathological characteristics. Breast cancer-derived MCF-7 and MDA-MB-231 cell lines were transfected with miR-18a and anti-miR-18a to evaluate the biological effects of this molecule. In addition, whole-transcriptome expression analysis was performed. Results High miR-18a expression in post-nCT residual tumors was found to be associated with a significantly worse overall survival [hazard ratio (HR): 2.80, 95% confidence interval (CI): 1.01–7.76] and a strong trend towards a poorer disease-free survival (HR: 2.44, 95% CI: 0.99–5.02) compared to low miR-18a expressing post-nCT residual tumors. Clinical and experimental data were found to be in conformity with the proliferative effects of miR-18a, which showed a significant correlation with Ki67 and MYBL2 expression, both in pre- and post-nCT tumors and in public databases. In vitro analysis of the role of miR-18a in breast cancer-derived cell lines showed that a high expression of miR-18a was associated with a low expression of the estrogen receptor (ER), a decreased sensitivity to tamoxifen and an enrichment in luminal B and endocrine resistance gene expression signatures. Conclusions From our data we conclude that post-nCT miR-18a expression in breast cancer serves as a negative prognostic marker, especially in luminal tumors. Clinical, in vitro and in silico data support the role of miR-18a in breast cancer cell proliferation and endocrine resistance and suggest its potential utility as a biomarker for additional adjuvant treatment in patients without a pathologic complete response to neoadjuvant therapy.

Project description:The Mexican axolotl provides a powerful model to investigate mechanisms of tissue regeneration. A recent chemical screen found that HDAC inhibitor romidepsin, administered for only 1-minute post amputation (1 MPA), blocks axolotl tail regeneration. Here, we tested the potential for cobalt chloride (CoCl2), a chemical stabilizer of HIF1a and inducer of hypoxia, to rescue romidepsin-inhibition of tail regeneration. Tail regeneration was partially rescued when embryos with amputated tails were co-treated with romidepsin and CoCl2. However, extending the CoCl2 dosage window either inhibited regeneration (CoCl2:0-30 MPA) or was lethal (CoCl2:0-24 hours post amputation; HPA). CoCl2:0-30 MPA caused tissue damage, tissue loss, and cell death at the distal tail tip, and blocked regeneration. In contrast, CoCl2 treatment of non-amputated embryos or CoCl2:60-90 MPA treatment of amputated embryos did not affect wound healing or inhibit tail regeneration. To further investigate the contrasting effects of CoCl2, microarray analysis was performed to identify differentially expressed genes at 3 HPA. CoCl2-romidepsin:1 MPA treatment significantly increased the expression of transcription factors associated with appendage regeneration, while CoCl2:0-30 MPA significantly increased expression of hemoglobin and platelet-specific transcripts, consistent with hemorrhage and an impaired hemostatic response. Also, CoCl2:0-30 MPA significantly increased expression of hypoxia inducible genes, including genes that encode Hif1a interacting proteins and heat shock proteins (HSP); in contrast, genes encoding TGFB signaling components were significantly downregulated. Using additional chemical inhibitors of tail regeneration, we identified transcriptional responses associated with HSP90 activity and TGFB signaling. Notably, geldanamcin decreased transcription of matrix metalloproteinases and sustained muscle-specific gene expression, suggesting a role for HSP90 in regulating extracellular matrix remodeling and muscle dedifferentiation. Our study shows the power of using chemical tools to precisely identify temporal windows within which critical biological processes are enacted during tissue regeneration.

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MiR-18a modulates gene transcription in breast cancer cells treated with Cobalt(II) chloride

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