Expression of microRNA-155 in thalassemic erythropoiesis

Study animals and bone marrow specimens

Wild type (WT) (β^m/m) and β^IVSII-654-thalassemic mice (Lewis et al., 1998) littermates under C57BL/6J background (twelve mice per each of the group) were obtained from the Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University, Thailand. The βIVSII-654-thalassemic C57Bl/6 mouse model demonstrated comparable features to thalassemia intermedia, including anemia, ineffective erythropoiesis and splenomegaly (Srinoun et al., 2009). Male or female 8-to-12-week-old β^IVSII-654-thalassemic and WT mice were housed 4–6 per cage per genotype. All twenty-four were given routine feeding with standard diet and water ad libitum. The temperature was 25 ± 2 °C and humidity was maintained at 55 ± 10%, with a 12-h light/dark cycle and clean conventional housing system. The mice were euthanized by carbon dioxide (CO₂) inhalation. No exclusion criteria were used.

After the mice were euthanized, bone marrows from the femur were collected by flushing the femurs with collection media containing 2% fetal bovine serum (FBS) in Iscove’s modified Dulbecco’s medium (IMDM) (GIBCO, Waltham, MA, USA) (Srinoun et al., 2009).

The protocol of this study was approved by the Ethics Review Board of the Institute of Science and Technology for Research and Development and the Institute of Molecular Biosciences, Mahidol University, Institute Animal Care and Use Committee (approval number MUSTA 2008-004 and COA.NO.IMB-ACUC 2021/011, respectively).

Purification of erythroblasts from mouse bone marrow

Erythroblast subpopulations from mouse bone marrow from 3 WT and 3 β-thalassemic mice were isolated by magnetic cell sorting using anti-mouse CD45 immunomagnetic beads (Miltenyi Biotec, Bergen-Gladbach, Germany) to separate white blood cells from erythroid cells. The cells were then separated into different stages of erythroid differentiation according to transferrin receptor levels using CD71(transferrin receptor) immunomagnetic beads. This resulted in two erythroblast subpopulations, CD45-CD71⁺Ter-119⁺ and CD45-CD71^-Ter-119⁺ in bone marrow. The purity and stage of erythroid differentiation were examined by staining with anti-mouse Ter-119 (red cell marker)-PE and anti-mouse CD71-fluorescein isothiocyanate (FITC) (BD Biosciences, San Jose, CA, USA) and analyzed using a FACSCalibur flow cytometer and CellQuest software (BD Biosciences).

Mouse bone marrow erythroid progenitor cell culture

Single cell suspensions of mouse bone marrow cells (WT n = 3, β-thalassemic mice n = 3) were suspended in collection media and Ter-119⁺ cells were depleted by negative selection using biotin-conjugated anti-mouse Ter-119 antibody (BioLegend, San Diego, CA, USA) and streptavidin microbeads (Miltenyi Biotec, Teterow, Germany). Ter-119⁻ cells were collected using a system of MACS cell separation (Miltenyi Biotec, Teterow, Germany) according to the manufacturer’s protocol. The Ter-119 negative cells were cultured in two-phase liquid culture (d’Arqom et al., 2021). Initially, the expansion phase was performed, Ter-119 negative cells were cultured in StemPro- 34 serum-free medium (Gibco, Waltham, MA, USA), supplemented with 1x nutrient supplement mix (Gibco), 1 µM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA, 2 mM L-glutamine (Gibco), 2 U/mL erythropoietin (Stemcell Technologies, Vancouver, Canada), 40 ng/mL mouse insulin-growth factor-1 (Stemcell Technologies), 180 ng/mL mouse stem cell factor (PeproTech, Rocky Hill, NJ), and 100 U/mL penicillin/ streptomycin (Gibco) at 2 × 10⁶ cells/mL in 48-well plates and incubated at 37 °C, 5% CO₂ for 5 days. Cells in the expansion phase were then cultured in conditioned media during the differentiation phase. Cells were cultured in Iscove’s modified Dulbecco’s medium (Gibco) supplemented with 15% FBS (Gibco), 2 mM L-glutamine, 2 U/mL erythropoietin, 200 µg/mL human holotransferrin (ProSpec-Tany TechnoGene Ltd., Ness-Ziona, Israel), 10 µg/mL insulin (Gibco), 1% bovine serum albumin (HiMedia Laboratories Pvt., Ltd. Maharashtra, INDIA), 0.1mM β-mercaptoethanol (Sigma-Aldrich) and, 100 U/mL penicillin/streptomycin at 37 °C, 5% CO₂ for 2 days.

Erythroid differentiation analysis

Erythroid cell morphology and cell marker during differentiation was evaluated by Wright–Giemsa staining and flow cytometry, respectively. The murine erythroid cell culture was cytospun and analyzed by observing the Wright-Giemsa-stained cells under a light microscope. For flow cytometry, cultured erythroblasts were stained with anti-mouse Ter-119-PE and anti-mouse CD71-FITC antibodies (BD Biosciences, Franklin Lakes, NJ, USA). Erythroid subpopulations were identified as previously described (Chen et al., 2009; Liu et al., 2006). Data of 100,000 events was acquired and analyzed using BD Accuri™ C6 Plus Flow Cytometry (BD Biosciences) and evaluated using FlowJo software v10 (FlowJo LLC, BD Bioscience).

Transfection of miR-155 mimic and anti-miR-155 inhibitor

During the expansion phase of the culture (day 0), mouse erythroid cells (5 × 10⁵ cells) were treated with 80 nM of either miR-155 miRNA mimic or negative mimic control (mirVana™ miRNA mimic; Applied Biosystems, Waltham, MA, USA) to increase miRNA expression (WT mice n = 3, β-thalassemic mice n = 3), or miR-155 inhibitor or negative inhibitor control (Anti-miR™ miRNA Inhibitor; Applied Biosystems) to inhibit miRNA expression (WT mice n = 3, β-thalassemic mice n = 3). Both, miRNA-mimic and -inhibitor were transfected into erythroid cells using Lipofectamine™ RNAiMAX (Invitrogen, Waltham, MA, USA). Erythroblast cells were collected on day1 and day2 (differentiation phase), and the expression of miR-155 and its mRNA target genes was analyzed using quantitative reverse transcription PCR (qRT-PCR). Erythroid cell differentiation was evaluated by flow cytometry.

Quantitative miRNA analysis by RT-qPCR

Total RNA was extracted from cells using a Hybrid-R™ miRNA Isolation Kit (Gene-All, Seoul, South Korea). The concentration of RNA was measured with a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) with a suitable A260/280 ratio of 1.8 to 2.2, and a suitable A260/230 ratio of >1.7 and stored at −20 C. miR-155 quantitation was performed by using TaqMan™ Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientific) and TaqMan ™ Universal Master Mix II with UNG (Thermo Fisher Scientific) according to the manufacturer’s protocol. Let7a and snoRNA202 were used as reference genes for in vivo and in vitro experiments, respectively. The expression of miRNA normalized on reference gene was calculated using 2^−ΔCt (comparative C_t) method by using WT or untreated as control. miRNA quantitation was analyzed using a LightCycler^® 480 PCR System (Roche Applied Science, Mannheim, Germany).

Analysis of putative mRNA expression targets of miR-155

The candidate mRNA targets of miR-155 (c-myc, bach1 and pu1) were validated using qRT-PCR. cDNA was synthesized from the total RNA and amplified in one-step qRT-PCR using the qPCRBIO SyGreen 1-step Go Lo-ROX Kit (PCR Biosystems Ltd., London, UK) with specific primers. The primers used in the qRT-PCR were as follows: c-myc: forward primer, 5′-ATGCCCCTCAACGTGAACTTC-3′; reverse primer, 5′-CCTCTTCTCCACAGACA CCAC-3′, bach1: forward primer 5′-GCCCGTATGCTTGAGTGAATT-3′, reverse primer 5′- CGTGAGAGCGAAATTATCCG-3′, pu1: forward primer, 5′-ATGTTACAGGC GTGCAAAATGG-3′, reverse primer, 5′-TGATCGCTATGGCTTTCTCCA-3′; Actb: forward primer, 5′-CGTTGACATCCGTAAAGACCTC-3′; reverse primer, 5′-AGCCACCGATCCACACAGA-3′. Firstly, reverse transcription step was performed at 45 °C for 10 min. Secondly, PCR amplification was performed with an initial denaturation at 95 °C for 2 min followed by 40 cycles of denaturation at 95 °C for 5 s, annealing and extension at 60 °C for 30 s, and melt curve stage. The experiments were performed in triplicates. The expression of mRNA target was normalized with β-actin (Actb) as an internal control and then performed using the 2^−ΔΔCt method. qRT-PCR was performed to amplify mRNA using a LightCycler^® 480 PCR System (Roche Molecular System).

Statistical analyses

Data were analyzed using Statistical Package for the Social Sciences version 23 (SPSS Inc., Chicago, IL, USA). Comparisons between parameters were conducted using the nonparametric Mann–Whitney U Test. Values of P <0.05 were accepted as statistically significant.

In vivo and in vitro upregulation of miR-155 in β-thalassemic mice erythropoiesis

IE has been reported in β-thalassemic mice (Chaichompoo et al., 2022a; Srinoun et al., 2009). To study miR-155 expression, erythroblasts from β-thalassemic mice bone marrow was separated into two erythroblast subpopulations: CD45-CD71⁺Ter-119⁺ and CD45-CD71⁻Ter-119⁺, represent early and late erythroblasts, respectively. The purity and stage of erythroid differentiation were determined by flow cytometry and Wright–Giemsa staining, respectively. The results demonstrated that the CD45-CD71⁺Ter-119⁺ fraction mainly contain basophilic and polychromatic erythroblasts, while the CD45-CD71⁻Ter-119⁺ fraction comprised mainly of orthochromatic erythroblasts and mature erythrocyte (Figs. 1A and 1B). Our results showed increased basophilic and polychromatic erythroblasts which represented by CD45-CD71⁺Ter-119⁺ fraction in β-thalassemic mice compared to WT (Figs. 1A and 1B). The expression levels of miR-155 in CD45-CD71⁺Ter-119⁺ and CD45-CD71⁻Ter-119⁺ fractions from WT and β-thalassemic mice were determined using qRT-PCR. Interestingly, miR-155 expression level in early erythroblast was significantly lower than that of late erythroblast in β-thalassemic mice. Moreover, miR-155 expression in two erythroblast subpopulations of β-thalassemic mice were significantly upregulated compared to that of wild type mice (Fig. 1C).

To investigate the expression of miR-155 during in vitro erythropoiesis, Ter-119-depleted erythroid progenitor cells isolated from the heterozygous β^IVSII-654-thalassemic mice bone marrow were cultured in a two-phase liquid culture system. In the expansion phase (day 0), cell proliferation did not differ between the thalassemic and WT mice. However, in the differentiation phase, the proliferation rate in cells derived from thalassemic mice increased to approximately 1.6-fold on day1 and 1.3-fold on day2 compared with that of the WT (Fig. 2A). These results correspond to increased proliferation of β-thalassemic erythroid cells due to IE. In the expansion phase, erythroblasts exhibited high CD71 and intermediate Ter-119 expression corresponding to proerythroblasts (region S1) in both WT and thalassemic mice (Figs. 2B and 2C). Although during differentiation phase, the main population of erythroblasts comprises early erythroblasts or proerythroblasts, the cells increasingly differentiated into basophilic erythroblasts (region S2), polychromatic erythroblasts (region S3), and orthochromatic erythroblasts (region S4) on day1 and day2, respectively (Figs. 2B and 2C). On day 2, the result revealed that polychromatic erythroblasts of thalassemic mice were significantly increased compared with those of WT mice (Fig. 2C). Erythroid cell differentiation was also evaluated by Wright–Giemsa staining, which showed that erythroblast morphology corresponded with erythroid marker outcomes. Moreover, our bone marrow culture conditions demonstrated that erythropoiesis occurred in an erythroblastic island niche. It consisted of a central macrophage encompassed by erythroblasts (Fig. 2D).

The expression level of miR-155 during in vitro erythropoiesis was significantly increased in β-thalassemic mice in differentiation phase, day 1 and day 2, compared that of wild type mice (Fig. 2E). Our data suggest that increased proliferation and differentiation of thalassemic mouse erythroblasts, in both in vivo and in vitro erythropoiesis, may be associated with miR-155 upregulation.

The effects of miR-155 expression on erythroid proliferation and differentiation

To evaluate the functional effects of miR-155 expression on erythroid proliferation and differentiation, gain-and loss-of-function was investigated. Transfection with miRNA-mimic significantly elevated miR-155 expression in both β-thalassemic and WT mice, compared with untreated and negative control groups (p = 0.001) (Fig. 3A). The upregulation of miR-155 in β-thalassemic mice correlated with significantly decreased the percentage of erythroblast representing proerythroblast (region S1). A significant increase in the percentage of basophilic erythroblasts (region S2) was observed with a gain in miR-155 expression. However, increased miR-155 levels did not affect erythroid differentiation in the WT group (Figs. 3C and 3D).

Loss-of-function during erythroid differentiation was assessed using an anti-miR-155 inhibitor. The results exhibited that transfection of cells with an anti-miR-155 inhibitor resulted in significant reduction in miR-155 expression compared to the untreated and negative control in both β-thalassemic (p = 0.05) and WT mice (p = 0.046) (Fig. 3B). In wild-type mice, the loss of miR-155 expression did not affect erythroid differentiation. However, a significant increase the percentage of early erythroblast (region S1) but decreased of the percentage of late erythroblast (region S2 and S3) was detected in β-thalassemic mice which were transfected with anti-miR-155 inhibitor, compared to that in untreated and negative inhibitor control-treated cells (Figs. 3E and 3F).

C-myc is a target of miR-155 in β-thalassemia erythropoiesis

To elucidate the probable functional association between miR-155 and cell differentiation, the predicted mRNA target genes of miR-155 were analyzed using TargetScan, MiRTarBase, and PicTar (Riolo et al., 2020; Undi, Kandi & Gutti, 2013). Three candidate mRNA targets, c-myc, bach-1, and pu-1, were selected from mRNA target prediction and previous publications showing their relationship with cell differentiation (Edalati Fathabad et al., 2017; Georgantas 3rd et al., 2007; Li et al., 2023; Palma et al., 2014; Penglong et al., 2023; Srinoun et al., 2017). Bach1 was selected from target of miR-155 in macrophage cells achieved from the bone marrow and spleen of β-thalassemic mice (Penglong et al., 2023). The level of c-myc, pu-1, and bach1 gene expression was determined in erythroblast cell derived from β-thalassemic and WT mice which were transfected with miR-155-mimic and -inhibitor. In the β-thalassemic and WT mice cells transfected with miR-155-mimic, the upregulation of miR-155 significantly decreased the expression of c-myc mRNA compared to that in untreated and negative miR-mimic-treated cells (p = 0.05) (Fig. 4A). However, there was no statistically significant difference in bach1 expression levels among the three groups in β-thalassemic and WT mice (p = 0.127 and p = 0.827, respectively) (Fig. 4B). The upregulation of miR-155 expression was significant increased pu-1 mRNA (p = 0.05) (Fig. 4C). Conversely, miR-155 downregulation correlated with the significant increase in c-myc mRNA expression in β-thalassemic and WT mice (p = 0.05) (Fig. 5A). Similar to the gain of miR-155 expression condition, there was no change in bach1 expression and also observed in pu-1 expression after anti-miR-155 inhibitor transfection in WT mice (p = 0.487 and p = 0.127, respectively) (Fig. 5B); however, the expression of pu-1 increased with miR-155 downregulation in β-thalassemic mice (p = 0.05) (Fig. 5C). Taken together, these results indicated that c-myc is a valid target gene of miR-155 that regulates erythroid differentiation.