Dataset Information

Pathway Analysis of Selected Circulating miRNAs in Plasma of Breast Cancer Patients: A Preliminary Study.

ABSTRACT: MicroRNAs in the circulation of breast cancer (BC) patients have great potential for the early diagnosis, treatment and monitoring of breast cancer. The aim of this preliminary study was to obtain the expression profile of selected miRNAs in the plasma of BC patients that could discriminate BC patients from healthy volunteers and may be useful in early detection of BC. Significantly deregulated miRNAs were evaluated by pathway analysis with the prediction of potential miRNA targets. The study enrolled plasma samples from 65 BC patients and 34 healthy volunteers. Selected miRNAs were screened in pilot testing by the real-time PCR (qPCR) method, and the most appropriate reference genes were selected for normalisation by the geNorm algorithm. In the final testing, we detected miR-99a, miR-130a, miR-484 and miR-1260a (p < 0.05) as significantly up-regulated in the plasma of BC patients. Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis revealed that all significantly deregulated miRNAs are involved in the Hippo and Transforming Growth Factor-beta (TGF-beta) signalling pathways. Our study confirmed a different profile of selected circulating miRNAs in the plasma of BC patients with an emphasis on some critical points in the analysis process.

SUBMITTER: Holubekova V

PROVIDER: S-EPMC7583045 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Pathway Analysis of Selected Circulating miRNAs in Plasma of Breast Cancer Patients: A Preliminary Study.

Holubekova Veronika V Kolkova Zuzana Z Grendar Marian M Brany Dusan D Dvorska Dana D Stastny Igor I Jagelkova Marianna M Zelinova Katarina K Samec Marek M Liskova Alena A Laucekova Zuzana Z Kudela Erik E Bobrovska Martina M Kalman Michal M Zubor Pavol P Dankova Zuzana Z

International journal of molecular sciences 20201002 19

MicroRNAs in the circulation of breast cancer (BC) patients have great potential for the early diagnosis, treatment and monitoring of breast cancer. The aim of this preliminary study was to obtain the expression profile of selected miRNAs in the plasma of BC patients that could discriminate BC patients from healthy volunteers and may be useful in early detection of BC. Significantly deregulated miRNAs were evaluated by pathway analysis with the prediction of potential miRNA targets. The study en ...[more]

PMID: 33023154

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Project description:Breast cancer is a common disease with distinct tumor subtypes which can be phenotypically characterized by estrogen receptor, progesterone receptor and HER2/neu receptor status. MiRNAs play regulatory roles in tumor initiation and progression. Altered miRNA expression has been demonstrated in a variety of cancer states to date presenting the potential for exploitation as cancer specific biomarkers. Blood presents an attractive medium biomarker discovery. This study investigated systemic miRNAs differentially expressed in Luminal A (ER+PR+HER2/neu-) breast cancer and their effectiveness as oncologic biomarkers in the clinical setting. Blood samples were prospectively collected from consenting patients with Luminal A breast cancer (n=10) and controls (n=10). RNA was extracted, reverse transcribed and subjected to microarray analysis (n=10 Luminal A; n=10 Control). Differentially expressed miRNAs were identified by artificial neural network (ANN) data-mining algorithms. Ethical approval was granted by the Clinical Research Ethics Committee, Galway Roscommon University Hospital Group. Written informed consent was obtained from all study participants. Blood samples were prospectively collected from 20 women; this included 10 consecutive women with a new diagnosis of Luminal A breast cancer and 10 healthy control participants. Luminal A status was confirmed with immunohistochemistry and fluorescence in situ hybridization (FISH). The blood samples for the healthy control group were collected from women residing in the same catchment area as the cancer cases. These women had no personal history of malignancy and no current inflammatory or infectious condition. Venous non-fasting whole blood samples were collected in BD vacutainers ® containing 18mg dipotassium EDTA anticoagulant (BD-Plymouth, PL6 7BP, UK). Total RNA was extracted from blood (1ml) using TRI Reagent BD (Molecular Research Centre, Inc). RNA concentration and integrity were evaluated by NanoDrop spectrophotometry (NanoDrop ND-1000 Technologies Inc., DE, USA) and Agilent Bioanalyzer RNA 6000 NanoChip Kit Series II (Agilent Technologies, Germany) analysis, respectively. MiRNA microarray profiling Expression profiling of circulating miRNAs was performed using TaqMan miRNA arrays and assays in accordance with the manufacturer’s instructions (Taqman Low Density Array Human microRNA Card A and Card B, Applied Biosystems, Foster City, CA, USA). In short, total RNA was reverse transcribed using Megaplex primer pool A (Applied Biosystems) which contained sequence-specific primers for 381 specific miRNAs plus 3 controls (pool A). An additional panel of 384 miRNAs (381 miRNAs and 3 controls, pool B) was performed on a subset of 4 cancers and 4 controls. Real-time quantitative PCR was performed for 667 miRNAs, using A and B microfluidic cards, each containing primers and probes for 381 specific miRNAs plus 3 controls and thermal-cycled on an Applied Biosystems 7900HT instrument. The TLDA cards contain three endogenous controls (RNU44, RNU48 and MammU6 which is repeated four times on each card). Each card also contains a negative control, an assay unrelated to any human species, ath-miR-159a.

Project description:BackgroundCirculating plasma miRNAs have been increasingly studied in the field of pituitary neuroendocrine tumor (PitNET) research. Our aim was to discover circulating plasma miRNAs species associated with growth hormone (GH) secreting PitNETs versus assess how the plasma levels of discovered miRNA candidates are impacted by SSA therapy and whether there is a difference in their levels between GH secreting PitNETs versus other PitNET types and healthy individuals.DesignWe compared plasma miRNA content and levels before and after surgery focusing on GH secreting PitNET patients. Selected miRNA candidates from our data and literature were then tested in a longitudinal manner in somatostatin analogues (SSA) treatment group. Additionally, we validated selected targets in an independent GH secreting PitNET group.MethodsmiRNA candidates were discovered using the whole miRNA sequencing approach and differential expression analysis. Selected miRNAs were then analyzed using real-time polymerase chain reaction (qPCR).ResultsWhole miRNA sequencing discovered a total of 16 differentially expressed miRNAs (DEMs) in GH secreting PitNET patients' plasma 24 hours after surgery and 19 DEMs between GH secreting PitNET patients' plasma and non-functioning (NF) PitNET patients' plasma. Seven miRNAs were selected for further testing of which miR-625-5p, miR-503-5p miR-181a-2-3p and miR-130b-3p showed a significant downregulation in plasma after 1 month of SSA treatment. mir-625-5p was found to be significantly downregulated in plasma of GH secreting PitNET patients vs. NF PitNET patients. miR-625-5p alongside miR-130b-3p were also found to be downregulated in GH PitNETs compared to healthy individuals.ConclusionsOur study suggests that expression of plasma miRNAs miR-625-5p, miR-503-5p miR-181a-2-3p and miR-130b-3p in GH secreting PitNETs is affected by SSA treatment. Additionally, miR-625-5p can distinguish GH secreting PitNETs from other PitNET types and healthy controls warranting further research on these miRNAs for treatment efficacy.

Dataset Information

Pathway Analysis of Selected Circulating miRNAs in Plasma of Breast Cancer Patients: A Preliminary Study.

Publications

Pathway Analysis of Selected Circulating miRNAs in Plasma of Breast Cancer Patients: A Preliminary Study.

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