Nuclear miR-223 affects chromatin organization and regulates Flotillin-1 gene expression in myeloid progenitors

Hematopoietic cell lineage-specification and fate are deeply regulated by epigenetic signals and microRNA (miRNA), the latest mainly mediating the post-transcriptional gene silencing of target mRNAs. However, recent studies indicate that miRNAs play also a role in the nucleus, where they can bind complementary DNA sequences at specific chromatin sites that induce epigenetic modifications silencing or activating gene promoters’ transcription. The nuclear activity of miRNAs paves the way for non-coding RNA function in somatic stem cell proliferation, lineage specification and differentiation. The alteration of miRNA nuclear function might induce neoplastic transformation.To address this issue, we carried out chromatin immunoprecipitation-coupled deep sequencing (ChIP-Seq) in HL60 cells undergoing Retinoic Acid (RA)-induced granulocytic differentiation. By using anti-Cy5 antibodies we determined at the whole genome level the genomic sequences complementarily bound by Cy5-labeled double-stranded oligonucleotides, mimicking the activity of endogenous miR-223 (miR-223-Cy5), or by scramble Cy5-miRNA (Control). Moreover, we investigated the chromatin status at these genomic regions with antibodies recognizing the activating (H3K4me3) and/or or repressing (H3K27me3) histone marks. Among the complementary sequences bound by miR-223, we selected an evolutionary conserved region in the Flotillin-1 (FLOT1) gene promoter. We found that this FLOT1 region is bound by a complex comprising miR-223, RISC component AGO1 and trithorax (TrxG) protein RBBP5 and enriched in H3K4me3 marks during myeloid differentiation. Flotillin-1 is an essential component of lipid-rafts associated protein, whose role in hematopoiesis is still largely unknown. Flotillin-1 mRNA and protein levels are increased in primary human CD34+ hematopoietic progenitors undergoing granulo-monocytic differentiation and during RA-induced differentiation of HL60 and NB4 cell lines. In HL60 and NB4 cell lines, the silencing of miR-223 significantly impaired the RA-induced upregulation of FLOT1 levels and www.mjhid.org Mediterr J Hematol Infect Dis 2024; 16; e2024035 Pag. 20 / 40granulocytopoiesis, whereas FLOT1 overexpression or silencing in myeloid progenitors, respectively enhanced or inhibited the expression of differentiation markers CD11b and CD14. We also found that FLOT1 overexpression drives CSF1R, a growth-factor receptor involved in myeloid differentiation, to Rab-4 endocytic vesicles, increasing receptor recycling to cell membrane after CSF1 stimulation. Consistently with the analysis of a large-scale AML datasets, FLOT1 mRNA expression is significantly downregulated in AML blasts compared to healthy bone marrow cells. The lowest expression of FLOT1 was identified in APLs. Data on FLOT1 mRNA and miR-223 expression from The Cancer Genome Atlas (TGCA) reported a consistency between their expression patterns and AML FAB classification; again, the lowest expression levels were identified in APL (M3). Overall, our findings suggest nuclear miR-223 as an epigenetic regulator of chromatin organization and of FLOT1 gene function during physiological and RA-induced myelopoiesis. Our observations are also consistent with the oncogenic role of aberrant expression of miR-223 and FLOT1 in AML and APL, thus opening novel insights into molecular mechanisms leading to leukemia and for the design of new intervention strategies.