Uncontrolled Diabetes Mellitus Has No Major Influence on the Platelet Transcriptome

Abstract

Background Diabetes mellitus (DM) has been associated with increased platelet reactivity as well as increased levels of platelet RNAs in plasma. Here, we sought to evaluate whether the platelet transcriptome is altered in the presence of uncontrolled DM. Methods Next-generation sequencing (NGS) was performed on platelet RNA for 5 patients with uncontrolled DM (HbA1c 9.0%) and 5 control patients (HbA1c 5.5%) with otherwise similar clinical characteristics. RNA was isolated from leucocyte-depleted platelet-rich plasma. Libraries of platelet RNAs were created separately for long RNAs after ribosomal depletion and for small RNAs from total RNA, followed by next-generation sequencing. Results Platelets in both groups demonstrated RNA expression profiles characterized by absence of leukocyte-specific transcripts, high expression of well-known platelet transcripts, and in total 6,343 consistently detectable transcripts. Extensive statistical bioinformatic analysis yielded 12 genes with consistently differential expression at a lenient FDR < 0.1, thereof 8 protein-coding genes and 2 genes with known expression in platelets (MACF1 and ITGB3BP). Three of the four differentially expressed noncoding genes were YRNAs (RNY1, RNY3, and RNY4) which were all downregulated in DM. 23 miRNAs were differentially expressed between the two groups. Of the 13 miRNAs with decreased expression in the diabetic group, 8 belonged to the DLK1–DIO3 gene region on chromosome 14q32.2. Conclusions In this study, uncontrolled DM had a remote impact on different components of the platelet transcriptome. Increased expression of MACF1, together with supporting predicted mRNA-miRNA interactions as well as reduced expression of RNYs in platelets, may reflect subclinical platelet activation in uncontrolled DM.

Transcriptome Analysis of Reticulated Platelets Reveals a Prothrombotic Profile

“…In the literature, available data on miRNA are extremely various in terms of number and abundance of transcripts. 66,67,69,79,81 However, the most abundant miRNAs in platelets (e.g., miR-223, let7 family) correlate with our results.…”

“…Platelet transcriptome analyses published so far were performed collecting platelets directly from PRP. 66,67,79 This approach allows to collect billions of platelet to acquire enough genetic material but does not allow to isolate subgroups of interest. With this study, we isolated platelets from PRP by flow cytometry sorting with both positive (CD41 marker and platelet size) and negative (DNA staining) selection and we isolated RPs from peripheral blood using the state of the art staining with TO.…”

“…Interestingly, the amount of miRNAs detected in our small-RNA sequencing is only slightly lower than reported previously in sequencing data of a billion platelets. 66,79,80 Whether this difference is due to better purity through the sorting process or to the lower total amount of extracted RNA, and consequently lower definition of platelet transcriptome, remains unclear. In the literature, available data on miRNA are extremely various in terms of number and abundance of transcripts.…”

A data-driven biocomputing pipeline with meta-analysis on high throughput transcriptomics to identify genome-wide miRNA markers associated with type 2 diabetes

“…A nested case-control study found significantly decreased hsa-miR-483-5p levels in the plasma of individuals with prediabetes which indicated its potential utility as a diagnostic predictor/circulating biomarker of β-cell function [45]. Significant downregulation of hsa-miR-539-5p was previously detected in the circulation of individuals with uncontrolled diabetes [46] while another study discovered its implications on both diabetes and the heart [47]. Down-regulation of both hsa-miR-1260a [48,49] and hsa--miR-4454 [49] in the circulation of individuals with T2D was also previously reported.…”

Impact of high platelet turnover on the platelet transcriptome: Results from platelet RNA-sequencing in patients with sepsis

“…While results from RNA-sequencing in platelets from septic patients have been reported recently [ 11 ], this is to our knowledge the first study to simultaneously analyse levels of immature platelets in sepsis. Also, by magnetic bead-based leukocyte depletion, we obtained highly purified RNA for RNA-sequencing as previously described [ 28 ]. For the first time, we introduced the use of unique molecular identifiers to eliminate PCR amplification biases which may be relevant in a transcriptome with reduced complexity as it is observed in platelets [ 10 ].…”