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MicroRNA-21 maintains HSC homeostasis
mals. It was found that miR-21 is involved in Gfi1-medi- ated modulation of myelopoiesis and regulates macrophage polarization and anti-inflammatory effects.18- 20 Besides, mice lacking miR-21 have reduced eosinophil levels in the peripheral blood (PB) and impaired eosinophil colony-forming capacity in the BM.21 Furthermore, anoth- er study revealed that miR-21 mediates hematopoietic suppression in myelodysplastic syndromes by targeting smad7, which is a negative regulator of the TGF-β/smad pathway.22 In particular, recent studies have defined miR- 21 as an onco-miRNA, which is frequently upregulated in many kinds of tumors, including hematologic malignan- cies.17,23 Taken together, these findings demonstrate that miR-21 plays significant roles in the hematopoietic sys- tem. On the other hand, miR-21 was reported to be impli- cated in the biology of several types of stem cells, includ- ing mesenchymal stem cells, cancer stem cells and embry- onic stem cells.17,24,25 However, the exact role of miR-21 in HSC populations is largely unclear.
In the present study, we first found that mouse HSC express high levels of miR-21 and that targeted deletion of miR-21 leads to abnormal hematopoiesis. Furthermore, miR-21 deficiency significantly impairs the quiescence and long-term reconstituting function of HSC. We demon- strated that miR-21 is involved in the maintenance of HSC homeostasis and function through modulation of the nuclear factor kappa B (NF-κB) pathway by regulating pro- grammed cell death 4 (PDCD4). Of note, miR-21 is also able to mitigate radiation-induced DNA damage in HSC. Collectively, these data indicate a key role for miR-21 in HSC biology and therefore broaden our knowledge of the physiological functions of miR-21.
Methods
Animals
Normal, wild-type (WT) C57BL/6J mice were purchased from the Institute of Zoology (Chinese Academy of Sciences, Beijing, China). miR-21flox/+ (miR-21fl/+) mice and Mx1-Cre mice were obtained from Shanghai Model Organisms Center (China). miR- 21fl/fl;Mx1-Cre mice were generated by crossing miR-21fl/fl mice with Mx1-Cre mice. Unless otherwise stated, miR-21 deletion was induced by intraperitoneally injecting 4- to 6-week old miR- 21fl/fl;Mx1-Cre+ mice with 250 mg of polyinosinic:polycytidylic acid (pIpC) (Sigma, St. Louis, MO, USA) every other day for a total of seven doses. Four weeks after pIpC treatment, these mice were used in subsequent experiments. Identically treated miR- 21fl/fl;Mx1-Cre- littermates served as controls. Congenic C57Bl/6 SJL CD45.1+ mice were kindly provided by Prof. Jinyong Wang (Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Science, Guangzhou, China). All animal experiments were approved by the Animal Care Committee of The Third Military Medical University (Chongqing, China).
Flow cytometry
Single-cell suspensions of BM, spleen and PB were prepared as we described previously.8,26 Detailed information about the anti- bodies used for flow cytometric analyses is provided in Online Supplementary Table S1. The cell cycle, apoptosis, in vivo 5-bro- modeoxyuridine incorporation and intracellular protein staining were analyzed as we reported elsewhere.8 Samples were detect- ed using a FACSverse or FACSCanto flow cytometer (BD Biosciences, San Jose, CA, USA) and data were analyzed using FlowJo10.0 software (TreeStar, San Carlos, CA, USA). Cell sort-
ing was performed using a FACSAriaII or FACSAriaIII sorter (BD Biosciences).
Lentiviral transduction
The recombinant lentivirus carrying the PDCD4 gene or specific short hairpin RNA (shRNA) against PDCD4, as well as the corre- sponding negative controls, were obtained from Hanbio Co. Ltd. (Shanghai, China). The lentivirus was then transduced into HSC as we described previously.8 Subsequently, transduced Lin- SCA1+c-KIT+ (LSK) cells (6×103) were purified with GFP expression through flow cytometry, and transplanted into 10.0 Gy-irradiated CD45.1+ WT recipients along with 5×105 CD45.1+ competitor BM cells. The sequence of sh-PDCD4 is as follows: 5’-GAGCTTG- TATATGAAGCCATTGTAA-3’.
Microarray analysis
Total RNA was isolated from freshly sorted miR-21fl/fl or miR- 21D/D LSK cells. After that, samples were hybridized on Mouse Clariom D arrays (Affymetrix, Santa Clara, CA, USA) in triplicate using the procedures described in the User Manuals. Raw data were then normalized using the robust multi-array average. Genes with a fold change in expression >1.4 and a P value <0.05 were defined as differentially expressed and are listed in Online Supplementary Table S3. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrich- ment were used to analyze the microarray data. The data have been deposited in the Gene Expression Omnibus database (acces- sion number GSE131603).
Statistical analysis
All results were analyzed using GraphPad Prism 6.0 software (La Jolla, CA, USA). Differences in data between two groups and multiple groups were analyzed by a two-tailed Student t-test and one-way analysis of variance, respectively. The survival rates were compared by a log-rank nonparametric test and displayed as Kaplan-Meier survival curves. Unless otherwise stated, data were obtained from at least three independent experiments. P values <0.05 were defined as statistically significant.
Results
miR-21 is enriched in hematopoietic stem cells and its conditional ablation skews hematopoietic differentiation
Previous miRNA expression profiling studies have shown that miR-21 is highly expressed in mouse BM.12,27,28 To evaluate the role of miR-21 in hematopoiesis, we first measured the expression of miR-21 in murine hematopoi- etic stem and progenitor cells (HSPC). It was found that the expression of miR-21 was relatively enriched in HSC compared with that in committed hematopoietic progen- itors (Figure 1A), which hints that miR-21 may play a potential role in HSC biology. We then generated a con- ditional knockout mouse model (miR-21fl/fl;Mx1-Cre) by crossing miR-21fl/fl mice with Mx1-Cre mice (Figure 1B). The deletion of miR-21 was induced in the hematopoietic compartment by seven injections of pIpC (hereafter, miR- 21fl/fl;Mx1-Cre- and miR-21fl/fl;Mx1-Cre+ mice are referred to as miR-21fl/fl and miR-21D/D mice, respectively), which was confirmed by genomic polymerase chain reaction (PCR) and quantitative real-time PCR (qRT-PCR) analysis (Figure 1C, D; Online Supplementary Figure S1D). However, we did not find significant differences in total cell number in the BM and spleen, or in the counts of
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