Distinct miRNA Signatures and Networks Discern Fetal from Adult Erythroid Differentiation and Primary from Immortalized Erythroid Cells

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs crucial for post-transcriptional and translational regulation of cellular and developmental pathways. The study of miRNAs in erythropoiesis elucidates underlying regulatory mechanisms and facilitates related diagnostic and therapy development. Here, we used DNA Nanoball (DNB) small RNA sequencing to comprehensively characterize miRNAs in human erythroid cell cultures. Based on primary human peripheral-blood-derived CD34+ (hCD34+) cells and two influential erythroid cell lines with adult and fetal hemoglobin expression patterns, HUDEP-2 and HUDEP-1, respectively, our study links differential miRNA expression to erythroid differentiation, cell type, and hemoglobin expression profile. Sequencing results validated by reverse-transcription quantitative PCR (RT-qPCR) of selected miRNAs indicate shared differentiation signatures in primary and immortalized cells, characterized by reduced overall miRNA expression and reciprocal expression increases for individual lineage-specific miRNAs in late-stage erythropoiesis. Despite the high similarity of same-stage hCD34+ and HUDEP-2 cells, differential expression of several miRNAs highlighted informative discrepancies between both cell types. Moreover, a comparison between HUDEP-2 and HUDEP-1 cells displayed changes in miRNAs, transcription factors (TFs), target genes, and pathways associated with globin switching. In resulting TF-miRNA co-regulatory networks, major therapeutically relevant regulators of globin expression were targeted by many co-expressed miRNAs, outlining intricate combinatorial miRNA regulation of globin expression in erythroid cells.

CRISPR Editing Enables Consequential Tag-Activated MicroRNA-Mediated Endogene Deactivation

“…It is therefore also possible that insertion of MRSs in another region of the 3 UTR or in the 5 UTR of BCL11A would have resulted in more efficient suppression of BCL11A. Another issue of import for any selected miRNA is whether its isolated exploitation for TAMED will be able to evoke clinically relevant suppression of a target gene given the "multipleto-multiple" nature of small RNA interactions with targets, in accordance with our own transcriptomic data [37]. Importantly, for the highly expressed miR-451a, our indicative clonal TAMED data show consequential suppression of BCL11A with ensuing γ-globin increases, and as a natural genetic phenomenon, 3 UTR mutations that abrogate natural or create illegitimate MRSs elsewhere have been shown to cause disease [90,91].…”

“…Among them, miR-451a is characterized by abundant late-erythroid expression and low expression in HSCs and non-erythroid lineages [39,[44][45][46][47][48], and according to miRDB is not predicted to naturally target BCL11A [49]. The high expression of miR-451a was recently confirmed in our transcriptomic analysis of erythroid differentiation cultures of adult-type primary CD34+ and human umbilical cord-blood-derived erythroid progenitor-2 (HUDEP-2) cells (NCBI GEO accession ID GSE165011A) [37], which established miR-451a for both cell models as the most highly expressed miRNA in all late-stage erythroid cells and as significantly upregulated during erythroid differentiation (mean log 2 fold change of 4.39). The same dataset was utilized to shortlist 73 additional differentially expressed erythromiRs in late-vs. early-erythroid samples based on their high and highly upregulated expression in adult late-erythroid cells, which would thus be suitable for alternative or multiplexed application of TAMED to target BCL11A or other erythroid disease modifiers (see Figure 1 for the top 20 differentially expressed miRNAs and Supplementary Table S1 for all 73 miRNAs).…”

“…S1 for all 73 miRNAs). 1 : mean normalized miRNA counts in late-erythroid samples and 2 : mean log 2 fold change of miRNA expression in late-vs. early-erythroid samples, both as calculated by DEGseq [37]. Representative of all seven UTR sites, potential insertion events are only indicated for 3 UTR 1, 593 nt upstream from the end of the BCL11A-XL long 3 UTR (3206 nt).…”

“…CD34+ cells were isolated from peripheral blood of healthy individuals after mononuclear cell isolation using a density gradient medium, in line with procedures originally published as Protocol C [98] and modified as previously described by us [37].…”

“…Given its independence from lineage-specific enhancer elements as targets, TAMED would allow modular use of MRSs and knockdown by cognate endogenous miRNAs for any combination of disease modifiers, so that any effect detected here for BCL11A would be of both conceptual and, at sufficient efficiency, potential therapeutic significance. To this end, the present study draws on insights from our own recently published transcriptomic analyses [37], in line with other studies and reviews of hematopoietic and erythroid miRNA expression [38][39][40][41][42][43], to shortlist miRNAs likely suitable for TAMED in erythroid cells, and employs CRISPR/Cas9 to insert erythroid-specific miRNA (erythromiR) MRSs into the 3 UTR of BCL11A, based on the canonical-NHEJ and HDR pathways for editing. Our study shows the potential of miRNAs as versatile tools for research and therapy, investigates and compares methods of tagging cells and uncovers difficulties and pitfalls of implementing TAMED.…”