MicroRNA 320a and Membrane Antigens as Tools to Evaluate the Pathophysiology of Platelets Stored in Blood Banks.

Our research group, through the analysis of miRNomes in platelet concentrates (PCs) stored in blood banks, identified and validated the miR-127 and miR-320a miRNAs as biomarkers of platelet storage lesions (PSLs) in PCs. In order to validate the miRNAs 127 and 320a methodologically, as PSL biomarkers in a large number of PC bags, we also evaluated important immunological markers involved in the platelet activation/aggregation process-the CD62P receptor (P-selectin), the surface glycoproteins (GP) IIb/IIIa, and the purinergic P2Y12 receptor-via flow cytometry. The miRNAs miR-127 and miR-320a were quantified by real-time quantitative PCR (RT-qPCR). To carry out this study, 500 collection tubes were used at the upper edge of the PC bags containing platelets. Each tube was divided into seven equal parts (totaling 3500 samples) for platelet analysis from 7 different storage days, where the 1st day represents the high-quality control, and the 7th day corresponds to the low-quality control of the platelets. After analyzing all parameters during storage days, it was concluded that the relative quantification of miR-320a below 0.50 and the CD62P receptor below 27.92% are reliable indicators of the absence of storage lesions in blood banks. We believe that the values found in the expression of the CD62P receptor legitimize the use of the miR-320a and miR-127 miRNAs to build a kit capable of accurately measuring whether the stored platelets are suitable for transfusion.

Role of miRNAs in Human T Cell Leukemia Virus Type 1 Induced T Cell Leukemia: A Literature Review and Bioinformatics Approach.

Human T cell leukemia virus type 1 (HTLV-1) was identified as the first pathogenic human retrovirus and is estimated to infect 5 to 10 million individuals worldwide. Unlike other retroviruses, there is no effective therapy to prevent the onset of the most alarming diseases caused by HTLV-1, and the more severe cases manifest as the malignant phenotype of adult T cell leukemia (ATL). MicroRNA (miRNA) dysfunction is a common feature of leukemogenesis, and it is no different in ATL cases. Therefore, we sought to analyze studies that reported deregulated miRNA expression in HTLV-1 infected cells and patients' samples to understand how this deregulation could induce malignancy. Through in silico analysis, we identified 12 miRNAs that stood out in the prediction of targets, and we performed functional annotation of the genes linked to these 12 miRNAs that appeared to have a major biological interaction. A total of 90 genes were enriched in 14 KEGG pathways with significant values, including TP53, WNT, MAPK, TGF-ß, and Ras signaling pathways. These miRNAs and gene interactions are discussed in further detail for elucidation of how they may act as probable drivers for ATL onset, and while our data provide solid starting points for comprehension of miRNAs' roles in HTLV-1 infection, continuous effort in oncologic research is still needed to improve our understanding of HTLV-1 induced leukemia.

Targeting aurora kinases as a potential prognostic and therapeutical biomarkers in pediatric acute lymphoblastic leukaemia.

Aurora kinases (AURKA and AURKB) are mitotic kinases with an important role in the regulation of several mitotic events, and in hematological malignancies, AURKA and AURKB hyperexpression are found in patients with cytogenetic abnormalities presenting a unfavorable prognosis. The aim of this study was evaluated the mRNA expression profile of pediatric Acute Lymphoblastic Leukaemia (ALL) patients and the efficacy of two AURKA and AURKB designed inhibitors (GW809897X and GW806742X) in a leukemia cell line as a potential novel therapy for ALL patients. Cellular experiments demonstrated that both inhibitors induced cell death with caspase activation and cell cycle arrest, however only the GW806742X inhibitor decreased with more efficacy AURKA and AURKB expression in K-562 leukemia cells. In ALL patients both AURKA and AURKB showed a significant overexpression, when compared to health controls. Moreover, AURKB expression level was significant higher than AURKA in patients, and predicted a poorer prognosis with significantly lower survival rates. No differences were found in AURKA and AURKB expression between gene fusions, immunophenotypic groups, white blood cells count, gender or age. In summary, the results in this study indicates that the AURKA and AURKB overexpression are important findings in pediatric ALL, and designed inhibitor, GW806742X tested in vitro were able to effectively inhibit the gene expression of both aurora kinases and induce apoptosis in K-562 cells, however our data clearly shown that AURKB proves to be a singular finding and potential prognostic biomarker that may be used as a promising therapeutic target to those patients.

Computational Identification and Characterization of New microRNAs in Human Platelets Stored in a Blood Bank.

Platelet concentrate (PC) transfusions are widely used to save the lives of patients who experience acute blood loss. MicroRNAs (miRNAs) comprise a class of molecules with a biological role which is relevant to the understanding of storage lesions in blood banks. We used a new approach to identify miRNAs in normal human platelet sRNA-Seq data from the GSE61856 repository. We identified a comprehensive miRNA expression profile, where we detected 20 of these transcripts potentially expressed in PCs stored for seven days, which had their expression levels analyzed with simulations of computational biology. Our results identified a new collection of miRNAs (miR-486-5p, miR-92a-3p, miR-103a-3p, miR-151a-3p, miR-181a-5p, and miR-221-3p) that showed a sensitivity expression pattern due to biological platelet changes during storage, confirmed by additional quantitative real-time polymerase chain reaction (qPCR) validation on 100 PC units from 500 healthy donors. We also identified that these miRNAs could transfer regulatory information on platelets, such as members of the let-7 family, by regulating the YOD1 gene, which is a deubiquitinating enzyme highly expressed in platelet hyperactivity. Our results also showed that the target genes of these miRNAs play important roles in signaling pathways, cell cycle, stress response, platelet activation and cancer. In summary, the miRNAs described in this study, have a promising application in transfusion medicine as potential biomarkers to also measure the quality and viability of the PC during storage in blood banks.

MicroRNAs as a Potential Quality Measurement Tool of Platelet Concentrate Stored in Blood Banks-A Review.

BACKGROUND: Platelet concentrate (PC) is one of the main products used in a therapeutic transfusion. This blood component requires special storage at blood banks, however, even under good storage conditions, modifications or degradations may occur and are known as platelet storage lesions. METHODS: This research was performed on scientific citation databases PubMed/Medline, ScienceDirect, and Web of Science, for publications containing platelet storage lesions. The results obtained mainly reveal the clinical applicability of miRNAs as biomarkers of storage injury and as useful tools for a problem affecting public and private health, the lack of PC bags in countries with few blood donors. The major studies listed in this review identified miRNAs associated with important platelet functions that are relevant in clinical practice as quality biomarkers of PC, such as miR-223, miR-126, miR-10a, miR-150, miR-16, miR-21, miR-326, miR-495, let-7b, let-7c, let-7e, miR-107, miR-10b, miR-145, miR-155, miR-17, miR-191, miR-197, miR-200b, miR-24, miR-331, miR-376. These miRNAs can be used in blood banks to identify platelet injury in PC bags. CONCLUSION: The studies described in this review relate the functions of miRNAs with molecular mechanisms that result in functional platelet differences, such as apoptosis. Thus, miRNA profiles can be used to measure the quality of storage PC for more than 5 days, identify bags with platelet injury, and distinguish those with functional platelets.

Differential Expression Profile of MicroRNAs During Prolonged Storage of Platelet Concentrates As a Quality Measurement Tool in Blood Banks.

Platelet concentrate (PC) is a key blood component, which even in good storage conditions, susceptible to cellular damage over time. Hence, blood banks discard unused PC bags after 5 days of storage. Biomarkers of PC quality are therefore highly sought after in blood bank governance. We used the data (Gene Expression Omnibus: GSE61856) generated with next-generation sequencing to examine the expression profiles of microRNAs (miRNAs) from PCs that were stored for 6 days in a blood bank, that is, 1 day longer than is normally stored PC. We identified the 14 most differentially expressed miRNAs by comparing a control PC on the first day of storage with the PCs on each of the subsequent 5 days of storage from day 1 to 6. In all, we identified nine miRNAs with the downregulated profile (miR-145-5p, miR-150-5p, miR-183-5p, miR-26a-5p, miR-331-3p, miR-338-5p, miR-451a, miR-501-3p, and miR-99b-5p) and five upregulated miRNAs (miR-1304-3p, miR-411-5p, miR-432-5p, miR-668-3p, and miR-939-5p). These miRNAs were validated by real-time quantitative PCR in 100 PC units. As each PC unit is composed of platelets of five individuals, the validation was thus performed in 500 individuals (250 men and 250 women, comprised 18-40 years old adults). The data were analyzed with hierarchical clustering and principal component analysis, which revealed the variation of mean relative expression and the instability of miRNAs half-life on the fourth day of PC storage, which coincides with time of onset of platelet storage lesions. These new observations can usefully inform future decision-making and governance in blood banks concerning PC quality.

The miRNA Profile of Platelets Stored in a Blood Bank and Its Relation to Cellular Damage from Storage.

Millions of blood products are transfused each year, and many lives are directly affected by transfusion. Platelet concentrate (PC) is one of the main products derived from blood. Even under good storage conditions, PC is likely to suffer cell damage. The shape of platelets changes after 5 to 7 days of storage at 22°C. Taking into consideration that some platelet proteins undergo changes in their shape and functionality during PC storage. Sixteen PC bags were collected and each PC bag tube was cut into six equal pieces to perform experiments with platelets from six different days of storage. Thus, on the first day of storage, 1/6 of the tube was used for miRNA extraction, and the remaining 5/6 was stored under the same conditions until extraction of miRNAs on each the following five days. Samples were sequenced on an Illumina Platform to demonstrate the most highly expressed miRNAs. Three miRNAs, mir127, mir191 and mir320a were validated by real-time quantitative PCR (RQ-PCR) in 100 PC bags tubes. Our method suggests, the use of the miRNAs mir127 and mir320a as biomarkers to assess the "validity period" of PC bags stored in blood banks for long periods. Thus, bags can be tested on the 5th day of storage for the relative expression levels of mir127 and mir320a. Thus, we highlight candidate miRNAs as biomarkers of storage damage that can be used as tools to evaluate the quality of stored PC. The use of miRNAs as biomarkers of damage is unprecedented and will contribute to improved quality of blood products for transfusions.