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. 2015 Feb 10;14:36.
doi: 10.1186/s12943-015-0301-9.

Deregulated microRNAs in Triple-Negative Breast Cancer Revealed by Deep Sequencing

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Free PMC article

Deregulated microRNAs in Triple-Negative Breast Cancer Revealed by Deep Sequencing

Yao-Yin Chang et al. Mol Cancer. .
Free PMC article

Abstract

Background: MicroRNAs (miRNAs) are short, non-coding RNA molecules that play critical roles in human malignancy. However, the regulatory characteristics of miRNAs in triple-negative breast cancer, a phenotype of breast cancer that does not express the genes for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, are still poorly understood.

Methods: In this study, miRNA expression profiles of 24 triple-negative breast cancers and 14 adjacent normal tissues were analyzed using deep sequencing technology. Expression levels of miRNA reads were normalized with the quantile-quantile scaling method. Deregulated miRNAs in triple-negative breast cancer were identified from the sequencing data using the Student's t-test. Quantitative reverse transcription PCR validations were carried out to examine miRNA expression levels. Potential target candidates of a miRNA were predicted using published target prediction algorithms. Luciferase reporter assay experiments were performed to verify a putative miRNA-target relationship. Validated molecular targets of the deregulated miRNAs were retrieved from curated databases and their associations with cancer progression were discussed.

Results: A novel 25-miRNA expression signature was found to effectively distinguish triple-negative breast cancers from surrounding normal tissues in a hierarchical clustering analysis. We documented the evidence of seven polycistronic miRNA clusters preferentially harboring deregulated miRNAs in triple-negative breast cancer. Two of these miRNA clusters (miR-143-145 at 5q32 and miR-497-195 at 17p13.1) were markedly down-regulated in triple-negative breast cancer, while the other five miRNA clusters (miR-17-92 at 13q31.3, miR-183-182 at 7q32.2, miR-200-429 at 1p36.33, miR-301b-130b at 22q11.21, and miR-532-502 at Xp11.23) were up-regulated in triple-negative breast cancer. Moreover, miR-130b-5p from the miR-301b-130b cluster was shown to directly repress the cyclin G2 (CCNG2) gene, a crucial cell cycle regulator, in triple-negative breast cancer cells. Luciferase reporter assays showed that miR-130b-5p-mediated repression of CCNG2 was dependent on the sequence of the 3'-untranslated region. The findings described in this study implicate a miR-130b-5p-CCNG2 axis that may be involved in the malignant progression of triple-negative breast cancer.

Conclusions: Our work delivers a clear picture of the global miRNA regulatory characteristics in triple-negative breast cancer and extends the current knowledge of microRNA regulatory network.

Figures

Figure 1
Figure 1
A 25-miRNA expression signature discriminating between triple-negative breast cancers and adjacent normal tissues. (A) The global miRNA expression portrait from each sample was investigated using principal component analysis. Each data point in the principal component plot is composed of gene expression data of 707 mature miRNAs from a sample. Triple-negative breast cancers are shown in red and adjacent normal tissues are shown in blue. (B) Hierarchical clustering analysis of triple-negative breast cancers (red) and adjacent normal tissues (green) was performed using the 25 differentially expressed miRNAs. Each row represents the normalized gene expression data of a miRNA and each column represents a tissue sample. The dendrogram depicts similarities in the gene expression profiles among the samples. (C) Triple-negative breast cancers (red) and adjacent normal tissues (blue) are clearly separated into two groups in the principal component analysis using the gene expression data of the 25 differentially expressed miRNAs.
Figure 2
Figure 2
RT-PCR validations of six differentially expressed miRNAs in the miR-532-502 cluster in triple-negative breast cancer. (A) A conceptual diagram illustrates the genomic loci harboring the 5p miRNA forms (brown) and 3p forms (orange) in the miR-532-502 cluster. The names of specific miRNA forms are provided under each locus. Relative expression levels of (B) miR-532-5p, (C) miR-188-3p, (D) miR-362-5p, (E) miR-501-3p, (F) miR-660-3p, and (G) miR-502-5p in triple-negative breast cancer tissues (n = 19) and adjacent normal tissues (n = 4) are shown. In each panel, paired tissue samples from the same patient are indicated with the same digits in the sample ID. The miRNA expression level in 557-N served as the calibrator control. All six miRNAs examined in the miR-532-502 cluster were markedly up-regulated in triple-negative breast cancer. Each bar represents the mean of triplicate measurements ± the SD.
Figure 3
Figure 3
RT-PCR validations of the differentially expressed miRNAs in the miR-301b-130b cluster and the miR-497-195 cluster. (A) A conceptual diagram illustrates the genomic loci harboring the 5p miRNA forms (brown) and 3p forms (orange) in the miR-301b-130b cluster. Relative expression levels of (B) miR-301b-5p, (C) miR-130b-5p, and (D) miR-130b-3p in triple-negative breast cancer tissues (n = 19) and adjacent normal tissues (n = 4) are shown. All three miRNAs in the miR-301b-130b cluster were markedly up-regulated in triple-negative breast cancer. (E) A conceptual diagram illustrates the genomic loci harboring the 5p miRNA forms (brown) in the miR-497-195 cluster. Relative expression levels of (F) miR-497-5p and (G) miR-195-5p in 23 samples are shown. Both miRNAs in the miR-497-195 cluster were down-regulated in triple-negative breast cancer. Each bar represents the mean of triplicate measurements ± the SD.
Figure 4
Figure 4
miR-130b-5p directly silences CCNG2 expression in triple-negative breast cancer cells. (A) Schematic diagram of the putative miR-130b-5p binding site in the 3′-UTR of CCNG2. The possible binding sequence of miR-130b-5p with the 3′-UTR of CCNG2 is shown in red. (B) Luciferase assays of CCNG2 in miR-130b-5p-overexpressed HEK-293 T cells. Two luciferase reporter constructs, Luc-CCNG2-UTR and Luc-CCNG2-UTR-mt3, were made to test whether miR-130b-5p directly targets the putative binding site in the 3′-UTR of CCNG2. The mutated nucleotides are indicated in red. Compared with the negative control, overexpression of miR-130b-5p was shown to significantly inhibit luciferase reporter activity with Luc-CCNG2-UTR, but not with Luc-CCNG2-UTR-mt3. Each bar represents the mean of triplicate measurements ± the SD; *p <0.05. (C) Endogenous expression analysis of CCNG2 in MDA-MB-231 cells transfected with lentiviral vectors containing miR-130b-5p precursor sequences. The endogenous expression levels of CCNG2 were significantly repressed after the forced expression of miR-130b-5p in MDA-MB-231 cells. (D) Comparisons of miR-130b-5p expression in normal breast tissues (n = 14), early-stage triple-negative breast cancers (n = 7), and advanced-stage triple-negative breast cancers (n = 17). MiRNA expression reads were normalized and analyzed in log10 scale. miR-130b-5p expression was significantly associated with both early-stage and advanced-stage triple-negative breast cancers.

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