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miR-127 controls HSC maintenance
are constantly required to replenish differentiated blood cells characterized by high turnover or to respond to peripheral injuries such as bleeding, a portion of HSC must awake from their dormant state and re-activate pro- liferation and differentiation programs.2 Thanks to their ability to correctly balance self-renewal and multi-potent differentiation the HSC pool size is maintained. However, the molecular pathways underlying this regulation are not completely understood, including the role of micro-RNA (miRNA). These are evolutionary conserved small non- coding RNA (ncRNA) that regulate mRNA stability and translation at the post-transcriptional level through non- perfect binding to target sequences.3
To study the potential role for miRNA in HSC self- renewal, we took advantage of the Pbx1 conditional knockout (Pbx1-cKO) mouse model. Pbx1 is a home- odomain transcription factor that positively regulates HSC quiescence.4 Its absence in post-natal HSC causes an exces- sive proliferation that ultimately leads to their exhaustion, indicating a profound self-renewal defect, and a premature myeloid differentiation at the expense of lymphoid differ- entiation.5 Therefore, the study of Pbx1-deficient HSC provides the opportunity to identify miRNA involved in the maintenance of HSC identity.
We employed Pbx1-cKO mice (and controls) to perform miRNA profiling of HSC and their immediate down- stream progeny multi-potent progenitors (MPP) character- ized by the absence of the Flk2 marker on their cell surface (Flk2-MPP, previously known as short-term HSC), repre- senting one of the very first differentiation steps from HSC, with similar multi-potent differentiation capacity but reduced self-renewal. This approach allowed the iden- tification of an HSC-specific miRNA, miR-127-3p. By modulating its activity through lentiviral vectors we demonstrate that miR-127 acts as a novel brake on differ- entiation that HSC employ to maintain their pool.
Methods
Mice
Tie2Cre+.Pbx1-/f and Mx1Cre+.Pbx1-/f mice have been described.4,6 Briefly, Tie2Cre+.Pbx1+/- and Mx1Cre+.Pbx1+/f mice were bred with Pbx1f/f mice to obtain Tie2Cre+.Pbx1-/f or MxCre+.Pbx1f/f Pbx1-cKO and their littermate controls.
miRNA profile and bioinformatic analysis
RNA was extracted with MirVana isolation kit (Ambion, ThermoFisher Scientific) from 1.4-2x103 HSC and 2-6x103 Flk2- MPPs (Table 1) sorted from the BM of Polyinosinic-polycytidylic acid [poly(I:C)] (InvivoGen)-treated Mx1Cre+Pbx1f/f and Mx1Cre-Pbx1f/f control mice (four experimental groups, 4-5 samples from individual mice/group). miRNA profiling was per- formed using the Megaplex TaqMan Assay system coupled with PreAmplification step (Rodent Pool A, Applied Biosystems, ThermoFisher Scientific). The expression level of 376 miRNA plus eight controls was obtained. Non-expressed miRNA (Ct level ≥35) were filtered out, whereas miRNA expressed in at least one of the four groups were further analyzed. Several nor- malization strategies were applied (quantile, median and endogenous normalization strategy) and quantile normalization was primarily chosen due to its smallest co-efficient of variation among replicates (cv=sdev/mean).7
Differential expression (DE) analysis was performed by Statistical Analysis of Microarray8 (SAM) (FDR with q-value <0.1).
Lentiviral constructs
Design and production of lentiviral vectors for stable ectopic miRNA-overexpression (OE vector) or functional downregulation (DR vector) were as previously described.9,10 Briefly, both vectors exploit the spleen focus forming virus (SFFV) promoter and couple miR-127-3p up- or downregulation with the expression of a co- transcribed fluorescence reporter protein (mOrange fluorescent protein, OFP, for OE vector and destabilized Green Fluorescence Protein, dGFP, for DR vector respectively). For the OE vector, a 274bp fragment containing murine pre-mir-127 was PCR-ampli- fied and cloned into the XhoI and MluI sites inside the EF1a intron of lentiviral transfer plasmid #1394 (SFFV.EFintron.OFP; described by Lechman et al.11). In the DR or ‘sponge’ vector, four tandem copies of an imperfectly complementary miR-127-3p target sequence were synthesized as described by Gentner et al.10 and cloned into the 3' untranslated region (XbaI-XmaI sites) of the #1031scrT (LV.SFFV.dGFP) lentiviral backbone.11 Third-generation lentiviral vector particles pseudotyped with VSV-G were generat- ed as described.12,13
Ethics
The study was approved by the Humanitas Clinical and Research Center - IRCCS Institutional Ethical Committee (prot. n. CE Humanitas, as per Ministerial Decree 127/14 of 8/2/2013).
Statistical analysis
Data are represented as mean±Standard Error (SE) when n>3. The significance of differences was determined by two-tailed Mann-Whitney unpaired test unless otherwise stated. P<0.05 was considered statistically significant (ns: not significant; * P<0.05; ** P<0.005; ***P<0.0005; ****P<0.0001). Statistical analyses were per- formed with GraphPad Prism (GraphPad Software).
Additional methods are presented in the Online Supplementary Methods.
Results
Pbx1-cKO mouse as a model to identify candidate miRNA regulating hematopoietic stem cell self-renewal Hematopoietic stem cells and Flk2–MPP were prospec- tively isolated from the BM of five Pbx1-cKO (Mx1Cre+.Pbx1f/f) and four control (Mx1Cre-.Pbx1f/f) individ- ual mice. The expression level of 376 different miRNA was obtained using a multiplexed Taq-Man-based real- time stem-loop PCR method14 and subjected to normaliza- tion, filtering and analysis (Figure 1A). A global analysis through unsupervised hierarchical clustering indicated a clear distinction between HSC and Flk2-MPP at the level of miRNA expression (Figure 1B), suggesting that miRNA regulate the first transition step in adult hematopoietic development. Within each group, Pbx1-deficient and con- trol cells clustered separately, linking miRNA to self- renewal impairment. Differential expression analysis indi- cated that 71 miRNA are differentially expressed (DE) dur- ing the physiological HSC-to-MPP transition (Figure 1C, right, and Online Supplementary Table S1). A similar analy- sis on Pbx1-deficient HSC revealed 48 miRNA DE com- pared to control HSC (Figure 1C, left), half of which (n=23) are concordantly DE in the HSC-to-MPP list (Online Supplementary Table S2), in accordance with the hypothe- sis that miRNA are involved in HSC self-renewal. This result is similar to that obtained by analyzing mRNA, with Pbx1-deficient HSC exhibiting a transcriptional profile typical of their immediate downstream progeny.4
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