Circulating small non coding RNAs and microparticles as potential markers of atherosclerotic plaque composition in type 1 diabetes

Background: Small non coding RNAs (sncRNAs) are endogenous short non coding molecules that regulate gene expression at post-translational level and are involved in several physiopathological processes. Circulating sncRNAs could be found free in biological fluids or loaded into extracellular vesicles, such as microparticles (MPs) in order to reach other tissues and amplify their signal. Next-generation sequencing (NGS) has become the main platform for biological research and biomarker discovery in the profiling of sncRNAs. Aim: The aim of this study was: 1) to set up a protocol using NGS technology for the identification and quantification of circulating sncRNAs involved in atherosclerotic plaque composition in type 1 diabetic patients (T1DM); 2) to characterize the phenotypes of circulating MPs derived from T1DM, associated with the plaque composition to evaluate the impact of these extracellular vesicles as carrier of specific small non coding RNAs, involved in these pathways. Material and Methods: Total RNA of 61 T1DM patients with fibrous (CFP; n 30) or calcified (CCP; n 31) carotid plaques was extracted from plasma samples, using a kit for biological fluids. For NGS sequencing, 25 CFP and 26 CCP were evaluated. The preparation of libraries was assessed using the Qiagen system. The sncRNA libraries pool was sequenced through the NGS sequencer MiSeq (Illumina), and the analysis performed by two bioinformatics tools (Partek Flow and CLC Genomics Workbench software). MPs derived from plasma of 40 T1DM patients with fibrous (CFP; n 20) or calcified (CCP; n 20) carotid plaques was assessed by centrifugation (40min x 14,000 rpm a 4°C) and characterized using flow cytometry (CytoFLEX, Beckman Coulter). Results: An unbiased and accurate sncRNome-wide quantification was obtained, detecting already known circulating sncRNAs (miRNAs, n 2632; piRNAs, n 3286; and tsRNAs, n 640). The bioinformatic analysis using two software on the already known 2632 miRNAs showed a different profile in T1DM with CCP compared to T1DM with CFP. Circulating level of several miRNA implicated in vascular remodeling and glucose metabolism were upregulated in patients with CCP, compared to CFP (miR-503-5p, miR-93-5p, miR-106b-5p and let-7d-5p) and downregulated (miR-451a, miR-10a-5p and miR-29b-3p) in patients with CCP, compared to CFP. We found that MPs released from endothelial cells and Platelets are enhanced in T1DM with vascular calcification (CCP) compare with T1DM with fibrous plaque (CFP); interestingly, a population of MPs derived from a niche of cells positive for CD34 and α-smooth muscle actin (αSMA) is also increased in CCP. Furthermore, the subgroup of MPs positive for calcification marker was significantly enhanced in patients with CCP in comparison to CFP patients with the main contribution given by CD34+ cells, suggesting a key role of these cells in the development of this vascular complication. Conclusions: In conclusion, our results demonstrate the power of NGS technology to identify a huge amount of circulating sncRNAs and to discover RNA molecules present in human plasma. The identification of new molecular biomarkers with this ultra-high throughput and sensitive technique (NGS) will help to go further insight specific pathophysiological processes, such as atherosclerotic plaque composition in diabetes, allowing a potentially more targeted therapeutic approach. Furthermore, we demonstrate that microparticles exhibit differential markers in the presence of vascular calcification suggesting a potential role as carrier of small molecules to amplify their signal.