Expression data from miR-223KO and miR-223WT bone marrow cells. Mus musculus

Total bone marrow (BM) from miR-223 knockout (mir-223-/-) and wildtype (miR-223+/+) mice 21 was extracted, prestimulated for 2 days. Then, the BM cells were simultaneously cotransduced with MSCV-Hoxa9-pgk-neomycin and a MSCV-Meis1-IRES-YFP by co-cultivation with irradiated (4,000 cGy) viral producers. HoxA9-Meis1 transduced cells were sorted for YFP expression and continuously selected with neomycin (1.4 mg/ml). Processing of the pre-miRNA through Dicer1 generates a miRNA duplex, consisting of a miRNA and miRNA* strand. Despite the general view that miRNA*s have no functional role, we further investigated miRNA* species in 10 deep sequencing libraries from mouse and human tissue. Comparing miRNA/miRNA* ratios across the miRNA sequence libraries revealed that 50% of the investigated miRNA duplexes exhibit a highly dominant strand. Conversely, 10% of miRNA duplexes show a comparable expression of both strands, while the remaining 40% exhibit variable ratios across the examined libraries as exemplified by miR-223/miR-223* in murine and human cell lines. Functional analyses revealed a regulatory role for miR-223* in myeloid progenitor cells, implying an active role for both arms of the miR-223 duplex. This was further underscored by the demonstration that miR-223 and miR-223* target the IGF1R/PIK3 axis and that high miR-223* levels associate with increased overall survival in acute myeloid leukemia (AML) patients. Thus, we found a supporting role for miR-223* in differentiating myeloid cells in normal as well as the leukemic cell state. The fact that the miR-223 duplex acts through both arms extends the complexity of miRNA-directed gene regulation of this myeloid key miRNA. Overall design: 2 biological replicates

我们提取了miR-223敲除（miR-223 knockout, miR-223-/-）与野生型（wildtype, miR-223+/+）小鼠的总骨髓（bone marrow, BM）样本[21]，并进行2天的预刺激培养。随后，通过与经4000 cGy辐照的病毒包装细胞共培养，将MSCV-Hoxa9-pgk-neomycin与MSCV-Meis1-IRES-YFP同时共转导至骨髓细胞中。对转导了HoxA9-Meis1的细胞进行YFP表达分选，并以1.4 mg/ml的新霉素进行持续筛选。

pre-miRNA经Dicer1加工后，可生成由成熟miRNA与miRNA*链组成的miRNA双链。尽管学界普遍认为miRNA*链无生物学功能，但我们针对小鼠与人类组织的10个深度测序文库中的miRNA*物种展开了深入研究。通过比对不同miRNA序列文库中的miRNA/miRNA*表达比值，我们发现50%的被检测miRNA双链仅存在一条优势表达链；另有10%的miRNA双链两条链的表达水平相当；剩余40%的miRNA双链在各检测文库中呈现不同的表达比值，以小鼠及人类细胞系中的miR-223/miR-223*为例。

功能分析结果显示，miR-223*在髓系祖细胞中具备调控作用，这表明miR-223双链的两条臂均具有活性功能。后续实验进一步证实，miR-223与miR-223*均可靶向IGF1R/PIK3轴，且miR-223*高表达与急性髓系白血病（acute myeloid leukemia, AML）患者的总生存期延长显著相关。综上，我们发现miR-223*在正常及白血病状态下的髓系细胞分化过程中均发挥辅助调控作用。miR-223双链通过两条臂发挥功能这一发现，进一步提升了该髓系关键miRNA所介导的基因调控的复杂性。

总体实验设计：2个生物学重复