RBCs as circulating repositories of miRNAs

MicroRNAs (miRNAs) are small non coding RNA involved in many physiological and pathological conditions by negatively regulating gene expression. In blood, miRNAs are usually carried within blood cells and EVs and/or associated with Ago2, resulting in the minimal RISC. Although RBCs were considered to lack nucleic acids, their miRNAs content has been recently demonstrated. Due to RBCs abundance in blood, they represent the major source of the same. RBCs have already been used into the clinics as drug delivery system, suggesting their role also in miRNAs delivery. In addition, stored RBCs undergo the so called “storage lesions” in which miR-451 and miR-196a levels could be dysregulated. Here we demonstrate that RBCs are particularly rich in stable miRNAs exploring the reasons of this stability. RBCs purified from fresh blood went through total RNA extraction and RT-PCR to detect miRNAs expression levels followed by miRNAs short-term stability studies. Afterwards, RBCs were subjected to a loading procedure without adding any molecule, obtaining unloaded (UL) RBCs to be compared with untreated (NT) ones. Moreover, RNA immunoprecipitation (RIP) method was optimised and performed to isolate and then quantify Ago2-bounded RNA. First, we found that miR-451 is highly expressed in RBCs, while miR-196a is less abundant. In the following analyses, miR-106b and U6 snRNA were initially chosen as endogenous controls, but they showed high variability. U6 snRNA demonstrated a higher loss in stability compared to other miRNAs. Thus, absolute quantification my means of standard curves were used instead. Despite a little initial loss, miRNAs were retained in UL and resulted stable over time. Further analysis suggested that miRNA stability is related to their binding with Ago2 proteins, which protect them from degradation or release. Therefore, this finding may significantly changes the RBCs exploitation as miRNAs delivery system, both in transfusion medicine and RBCs-based cell therapies.