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Polycistronic clustering characteristics of deregulated microRNA genes in triple-negative breast cancer revealed by deep sequencing

ABSTRACT: Twenty-four triple-negative breast cancer and 14 adjacent normal tissues were collected from breast cancer patients during surgeries at National Taiwan University Hospital (NTUH, Taipei, Taiwan). All triple-negative breast cancer samples were invasive ductal carcinomas (IDC) and were negative in immunohistochemical statuses of ER, PR, and HER2 receptors, as confirmed by professional pathologists. Treatment procedure of all patients followed the National Comprehensive Cancer Network (NCCN) guideline. All samples were neoadjuvant-free and were collected before systemic chemotherapy treatments. Written informed consent was obtained from all patients who participated in this study. Using human tissues for research in this study was approved by the institutional review board at NTUH. A novel set of 25-miRNA signature identified in this study was able to effectively distinguish between triple-negative breast cancer and adjacent normal tissues. Moreover, we documented the first evidence of seven polycistronic miRNA clusters preferentially harboring deregulated miRNA genes in triple-negative breast cancer. In the present study, a panel of 24 triple-negative breast cancer and 14 adjacent normal tissue samples were examined for the presence of deregulated miRNA genes using the high-throughput sequencing technology. Total RNA was extracted from the triple-negative breast cancer and adjacent normal samples for preparation of small RNA libraries. Each small RNA library was constructed from total RNA of each sample using the SOLiD Total RNA-Seq Kit (Applied Biosystems, Foster City, CA, USA). Upon completion of polymerase chain reaction (PCR) amplification, small RNA libraries were purified using the SOLiD Library Micro Column Purification Kit (Applied Biosystems) and hybridized to the template beads using the SOLiD EZ bead system (Applied Biosystems). The template beads were amplified and deposited onto subtract for ligation sequencing by SOLiD 4 System (Applied Biosystems).

ORGANISM(S): Homo sapiens

SUBMITTER: Chang King-Jen

PROVIDER: S-ECPF-GEOD-40049 | biostudies-other |

REPOSITORIES: biostudies-other

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Project description:Twenty-four triple-negative breast cancer and 14 adjacent normal tissues were collected from breast cancer patients during surgeries at National Taiwan University Hospital (NTUH, Taipei, Taiwan). All triple-negative breast cancer samples were invasive ductal carcinomas (IDC) and were negative in immunohistochemical statuses of ER, PR, and HER2 receptors, as confirmed by professional pathologists. Treatment procedure of all patients followed the National Comprehensive Cancer Network (NCCN) guideline. All samples were neoadjuvant-free and were collected before systemic chemotherapy treatments. Written informed consent was obtained from all patients who participated in this study. Using human tissues for research in this study was approved by the institutional review board at NTUH. A novel set of 25-miRNA signature identified in this study was able to effectively distinguish between triple-negative breast cancer and adjacent normal tissues. Moreover, we documented the first evidence of seven polycistronic miRNA clusters preferentially harboring deregulated miRNA genes in triple-negative breast cancer. In the present study, a panel of 24 triple-negative breast cancer and 14 adjacent normal tissue samples were examined for the presence of deregulated miRNA genes using the high-throughput sequencing technology. Total RNA was extracted from the triple-negative breast cancer and adjacent normal samples for preparation of small RNA libraries. Each small RNA library was constructed from total RNA of each sample using the SOLiD Total RNA-Seq Kit (Applied Biosystems, Foster City, CA, USA). Upon completion of polymerase chain reaction (PCR) amplification, small RNA libraries were purified using the SOLiD Library Micro Column Purification Kit (Applied Biosystems) and hybridized to the template beads using the SOLiD EZ bead system (Applied Biosystems). The template beads were amplified and deposited onto subtract for ligation sequencing by SOLiD 4 System (Applied Biosystems).

Project description:BackgroundMicroRNAs (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.MethodsIn 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.ResultsA 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.ConclusionsOur 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.

Project description:Tumor-initiating cells with reprogramming plasticity and/or de-differentiation attributes have been thought to initiate primary tumor development as well as to regenerate secondary tumors in metastatic organs; however, the molecular mechanisms are not fully understood. We previously found that breast tumor-initiating cell marker, CD44, directs multicellular aggregation and cluster formation of circulating tumor cells (CTCs), which further enhance stemness and survival of such cells, enabling metastatic colonization to the lungs. To further elucidate the molecular network underlying CTC cluster formation, we performed global proteomic profiling and discovered that the tetraspanin protein CD81, which is normally enriched in exosomes (small extracellular vesicles), is a new driver of cancer initiation and metastasis as a facilitator and target of CD44. Loss of CD81 compromises tumorigenicity and mammosphere formation of triple negative breast cancer (TNBC) cells. Assisted by machine learning-based algorithms and mutagenesis approach, we found that CD81 interacts with CD44 on the cellular membrane through their extracellular regions. Notably, genetic knockout of CD44 or CD81 results in loss of both CD81 and CD44 in secreted exosomes, a state which abolishes exosome-induced self-renewal of recipient cells, such as mammosphere formation. In addition, RNA sequencing analysis showed that CD81 knockdown up-regulates expression of a cell differentiation marker, SEMA7a, whose down-regulation partially rescues mammosphere formation inhibition by CD81 depletion. Clinically, CD81 expression was observed in >80% of CTCs and specifically enriched and co-expressed along with CD44 in clustered CTCs of breast cancer patients. Mimicing the phenotypes of CD44 deficiency, loss of CD81 also inhibited tumor cell aggregation and lung metastasis of TNBC in both human and mouse tumor models, supporting the clinical significance of CD81 in association with patient outcomes. Our study highlights a new driving role of CD81 in cancer exosome-induced stemness, clustered CTCs, and metastasis initiation of TNBC, reported for the first time to our knowledge.

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Polycistronic clustering characteristics of deregulated microRNA genes in triple-negative breast cancer revealed by deep sequencing

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