High-Throughput Mirna Analysis Suggests Pro-Inflammatory Profile in Sickle Cell Disease
Authors: Cannon Matthew , Glass Sarah , Smith Sidney , Heinlein Melanie , Lapalombella Rosa , Desai Payal C 
Published: October 2019

Abstract
BACKGROUND: Mature circulating red blood cells, though devoid of a nucleus, have been shown to contain an abundance of miRNAs. Further, it has been shown that sickle cell patient-derived RBCs have a dramatic difference in miRNA content than normal RBCs. Given that a range of miRNAs are involved in the regulation of immunity, including the release of inflammatory mediators, we hypothesize that miRNAs enriched in circulating red blood cells function to prolong the inflammatory state in sickle cell disease. Further, we hypothesize that these miRNAs can be used as biomarkers for use in the clinic to predict crisis and differentiate acute versus chronic pain. Exploring this miRNA enrichment in circulating red blood cells in sickle cell patients will provide practical insight for the inflammation state and will inform characteristics of patients who may need greater care in the clinic.
METHODS: Twenty steady state patients were recruited and categorized according to their chronic pain status and crisis frequency per year. Whole blood was drawn during routine visits to the OSU Wexner Medical Center Hematology Clinic. Additionally, whole blood was drawn from five patients either in acute pain crisis (recruited prior to crisis) or within a few days of crisis. Samples were subject to double gradient centrifugation and red cells were resuspended in Trizol and cryopreserved. MiRNAs were isolated from red cell Trizol suspensions using a commercial isolation kit (QIAGEN Cat#217004). Isolated miRNAs were then subject to a NanoString Human miR (v3) expression assay. Differential expression analysis was conducted to compare miRNAs with at least 1.5 fold difference (p = 0.05) between steady state and acute crisis. Target prediction and GO ontology analysis was performed for statistically significant miRNAs using DIANA Tools mirPath v3. Follow-up qPCRs were performed using TaqMan Advanced miRNA cDNA Synthesis Kit (Cat#A28007) and TaqMan Advanced miRNA Assays (Cat#A25576) to validate the decreased expression of miRNAs. Additional qPCRs were performed using TaqMan Gene Expression Assays (Cat#4331182) to investigate mRNA regulatory effects of significant miRNAs in the total red cell population. Western blots were also performed to investigate regulatory effects of these miRNAs at the protein level.
RESULTS & CONCLUSION: Comparison of RBC miRNA profiles from patients during acute crisis to those in steady state shows several significantly decreased (>1.5 fold) miRNAs in crisis. Among these miRs we have found previously uncharacterized miRNAs, hsa-miR-2116-5p and hsa-miR-302d-3p. DIANA tools miRNA analysis software predicts these miRNAs to be involved in regulation of cell-to-cell adhesion pathways through gene transcripts such as Protocadherin Beta 6 (PCDHB6) and Neural Cell Adhesion Molecule 2 (NCAM2). Interestingly, inspection of miRNA predicted targets that fall under significant GO terms also predicts several individual miRNAs to regulate inflammatory response and nociceptive signaling gene transcripts like A20 (TNFAIP3) and Cathepsin S (CTSS). Validation of these miRNAs was performed via qPCR for 5 out of the 6 significantly decreased miRNAs. Of the 5 miRNAs tested, hsa-miR-2116-5p, hsa-miR-302d-3p, and hsa-miR-1246 were validated as having decreased expression in acute crisis patients compared to steady state. qPCRs were then performed to probe for miRNA based regulation of top predicted target mRNA transcripts. Both CTSS and TNFAIP3 showed increased expression of mRNA transcripts in acute crisis patient red cells as compared to steady state. Next, western blot analysis was performed on red cell protein lysate. Interestingly, this analysis revealed a pattern in activated CTSS expression that was independent of acute crisis. Steady state patients reporting chronic pain showed increased activated CTSS compared to those without chronic pain. Activated CTSS was not found in red cell lysates from three normal, non-SCD donors. Taken together, these results suggest that red blood cells may play a larger role in inflammation and pain responses in sickle cell disease than previously thought. Further these results suggest activated CTSS as a potential biomarker for differentiating chronic pain in patients. Follow-up studies are underway to further stratify and investigate these findings.

Disclosures
Desai: University of Pittsburgh: Research Funding; Ironwood: Other: Adjudication Committee; NIH: Research Funding; FDA: Research Funding; Selexy/Novartis: Research Funding; Pfizer: Research Funding.