Inducible overexpression of zebrafish microRNA-722 suppresses chemotaxis of human neutrophil like cells

Highlights

• Inducible co-expression of a protein coding gene and a microRNA in HL-60 cells.

• dre-miR-722 over-expression suppresses signaling in differentiated HL-60 cells.

• dre-miR-722 directly suppresses the expression of human RAC2 gene.

• dre-miR-722 suppresses chemotaxis and oxidative burst in HL-60 cells.

Abstract

Neutrophil migration is essential for battling against infections but also drives chronic inflammation. Since primary neutrophils are terminally differentiated and not genetically tractable, leukemia cells such as HL-60 are differentiated into neutrophil-like cells to study mechanisms underlying neutrophil migration. However, constitutive overexpression or inhibition in this cell line does not allow the characterization of the genes that affect the differentiation process. Here we apply the tet-on system to induce the expression of a zebrafish microRNA, dre-miR-722, in differentiated HL-60. Overexpression of miR-722 reduced the mRNA level of genes in the chemotaxis and inflammation pathways, including Ras-Related C3 Botulinum Toxin Substrate 2 (RAC2). Consistently, polarization of the actin cytoskeleton, cell migration and generation of the reactive oxygen species are significantly inhibited upon induced miR-722 overexpression. Together, zebrafish miR-722 is a suppressor for migration and signaling in human neutrophil like cells.

Introduction

The neutrophil is the most abundant white blood cell in the circulation and a significant regulator of inflammation. While essential for battling against pathogens, neutrophil activation drives immunopathology in numerous human diseases, including organ transplantation, sepsis, rheumatoid arthritis, diabetes, neurodegenerative disease and cancer (Borregaard, 2010; Nathan, 2006; Soehnlein et al., 2017), although the link of some diseases to the innate immune system is not intuitive. Besides killing pathogens, they communicate with other cells to shape the inflammatory response. For example, neutrophils initiate inflammation by scanning platelets (Sreeramkumar et al., 2014), migrating away from the initial activation site to disseminate inflammation to the lung (Woodfin et al., 2011), priming macrophages by providing DNA in the forms of extracellular traps (Warnatsch et al., 2015) and can directly present antigens to activate T cells (Abi Abdallah et al., 2011; Lim et al., 2015).

Manipulating neutrophil migration and activation is implicated in managing chronic inflammation (Kolaczkowska and Kubes, 2013; Soehnlein et al., 2017). The current challenge in the field is that primary neutrophils are terminally differentiated with a very short life span ex vivo, which excludes the feasibility of genetic manipulation for functional characterization. To model neutrophils, human promyelocytic leukemia HL-60 cells (Pedruzzi et al., 2002) and NB4 cells (Lanotte et al., 1991) are differentiated in culture for 5–7 days into neutrophils-like cells. Gene transduction approaches using either the lenti- or retro-virus are successful in these cell lines, providing a genetically tractable system. However, due to the cell differentiation process, extensive characterization is required to separate the effect of the target genes on cell differentiation and mature cell function. Inducible expression using tet-on (gene expression activated by doxycycline) is widely applied in cancer research and other cell lines. However this technique has not been applied to neutrophil precursor cells.

MicroRNAs (MiRNA) are evolutionarily conserved, non-coding RNAs of ˜22 nucleotides that post-transcriptionally regulate gene expression (Fabian and Sonenberg, 2012). miRNAs are master regulators that can simultaneously suppress hundreds of genes and regulate numerous cellular processes and human diseases. miRNA profiles are distinct in human peripheral blood neutrophils (Gantier, 2013; Landgraf et al., 2007; Ward et al., 2011) and activated tissue infiltrating neutrophils (Larsen et al., 2013), suggesting that they are regulated by the inflammation process or tissue environment. On the other hand, only a few miRNAs are functionally characterized in neutrophils (Gurol et al., 2016). In HL-60 cells, miRNA expression changes during cell differentiation (Jian et al., 2011; Kasashima et al., 2004; Pizzimenti et al., 2009) and after radiation (Liamina et al., 2017) or resveratrol treatment (Ergin et al., 2015). Indeed, multiple miRNAs regulate HL-60 growth, differentiation and survival in vitro (Bousquet et al., 2008; Chen et al., 2010; Huang et al., 2015; Jian et al., 2011; Kawasaki and Taira, 2004; Lin et al., 2015; Sharifi et al., 2014; Shen et al., 2016; Wang et al., 2016; Wang et al., 2012). On the other hand, reports on how miRNAs regulate differentiated cell function such as migration is scarce. Introduction of synthetic miR-155 and miR-34 mimics into differentiated HL-60 suppressed cell migration but not differentiation (Cao et al., 2017), whereas depleting miR-155, on the other hand, induced cell differentiation and apoptosis (Liang et al., 2017).

In zebrafish, we have identified a miRNA, miR-722, that when overexpressed in neutrophils, reduces neutrophil chemotaxis and protects the organism from both sterile and non-sterile inflammatory assaults (Hsu et al., 2017). A hematopoietic specific isoform of the small GTPase, rac2, was identified as a direct target of miR-722. Here, we used the tet-on technique to induce the overexpression of miR-722 in differentiated HL-60 cells and uncovered a similar suppressive function of miR-722 in human neutrophils.