MicroRNA-Ago2 Complex in Mature Human Red Blood Cells.
Authors: Umemura Tsukuru , Tanaka Yuka , Fujisaki Megumi , Masaki Shizuka , Shiotsu Hiromichi , Srinoun Kanitta , Fucharoen Suthat , Abe Yasunobu , Ichihara Hisako , Toh Hiroyuki , Hisaeda Hajime 
Published: October 2019

Abstract
Abstract 4042
Poster Board III-977

Background
MicroRNA (miRNA) is noncoding short RNA which plays important roles for cell death, proliferation, development and differentiation. MiRNA binds to 3' UTR region of target messenger RNA(mRNA), and negatively regulates gene expressions by inducing instability of mRNAs or translation block. This function of miRNA depends on the presence of cytoplasmic protein complex, RNA induced silencing complex(RISC). A main component of RISC is argonoute2(Ago2), of which deficiency or knockout results in severe cellular dysfunction. We and several groups reported erythroid-specific expressions of miRNAs(Masaki et al., BBRC 346:509-514, 2007). Among those miRNAs, miR-451 is especially specific to erythroid cells. Knockout of miR-451 results in reduction of red blood cell(RBC) production, and a high amount of miR-451 is contained in RBCs which are enucleated, and previously seem to be genetically inactive. Roles of miRNAs in mature RBCs for the biology of RBC are entirely unclear. In this study, we analyzed amounts of miRNA-Ago2 complexes in normal human RBCs.

Materials & Methods
Circulating blood cells were obtained from 5 normal subjects, and fractionated using density centrifugation and ultracentrifugation methods. RNA was isolated directly from each cell fraction, or isolated after immunoprecipitation by anti-Ago2 antibody(Wako, Osaka, Japan). MiRNA was measured by reverse transcriptase-based quantitative real time polymerase chain reaction(RT-qPCR), using microRNA assay kits(ABI).

Results
Packed RBCs contained miR-451 at the concentrations of 10,000 folds higher than granulocytes and miR-16 at the same level, whereas miR-155, -152, -221, -223 were contained at very low levels. After ultracentrifugation of circulating RBCs, packed RBCs were fractionated into 4 fractions:buffy coat cells(BFC), upper, middle, lower fractions of packed RBC(BFC, UF, MF, LF, respectively). Numbers of leukocytes were 21.9 and 0.88 per 104 RBCs in BFC and UF, respectively. They were low in MF and LF, 0.20 and 0.10. Coexisting reticulocytes in each fractions were 15.3, 5.50, 3.75 and 1.75 per 106 RBCs in BFC, UF, MF, LF, respectively. We used let-7a as an internal control for RT-qPCR assays, since expression of let-7a was relatively constant between 4 fractions. Analysis of total RNA isolated directly from each fraction showed constantly high expression of miR-16 and -451 in RBCs from each fraction. Analysis of immunoprecipitated miRNAs showed that the almost same amounts of miR-16 and -451 were present as miRNA-Ago2 complex in RBCs from the lower fraction(mature RBCs).

Discussion & Conclusion
We report, for the first time, the presence of miRNA-RISC complex in mature RBCs. Ratio of miRNA bound to Ago2 in total miRNAs is depend on species of miRNA. Our observation showed that almost all miRNA in mature RBCs is present as a miRNA-RISC complex. This finding clearly suggests that miRNAs in mature RBCs are active molecules, and also suggests that enucleated mature RBCs are still using epigenetic controlling system for some biological purpose. The well-known target gene of miR-16 is BCL2. There is a possibility that miR-16 negatively regulates expression of immortalize gene, BCL2, resulting in suppression of proliferation, and in keeping a differentiated state. On the other hand, the target gene of miR-451 is not well determined. Experiments using Zebra fishes showed knockout of miR-451 induced severe reduction of RBC production, suggesting that miR-451 is one of key molecules for erythropoiesis. In conclusion, our observations suggest that miRNA-Ago2 complexes in mature RBCs may be playing roles for homeostasis of normal erythropoiesis, and the analysis of miRNAs-Ago2 complex in RBCs is relevant for understanding normal and pathological erythropoiesis.

Disclosures:
No relevant conflicts of interest to declare.