Circular RNA hsa_circ_0008726 Targets the hsa-miR-206-3p/KLF4 Axis to Modulate 4,4'-Methylene Diphenyl Diisocyanate-Glutathione Conjugate-Induced Chemokine Transcription in Macrophages

Abstract

Exposure to 4,4'-methylene diphenyl diisocyanate (MDI) in the workplace may lead to the development of occupational asthma (OA). However, the specific mechanism(s) by which MDI induces OA are poorly understood. Previous reports have demonstrated that MDI and MDI-glutathione (GSH) conjugate exposure downregulates endogenous human/murine (hsa/mmu)-microRNA(miR)-206-3p, resulting in the activation of mmu/hsa-miR-206-3p-regulated signaling pathways in macrophages. Circular RNAs (circRNAs) regulate many important biological processes by targeting endogenous miRs; however, whether MDI/MDI-GSH exposure may influence circRNA expressions is unknown. Several circRNAs have been identified that regulate hsa-miR-206-3p. We hypothesize that MDI-GSH conjugate exposure induces endogenous circRNA(s) to regulate hsa-miR-206-3p in macrophages. The expression of candidate hsa-miR-206-3p-binding circRNAs was determined from MDI-GSH conjugate-treated differentiated THP-1 macrophages using RT-qPCR. MDI-GSH exposures induced hsa_circ_0008726 and its host gene transcript DNAJB6, whereas other circRNA(s) examined were either not detected or unchanged. RNA-induced silencing complex-immunoprecipitation (RISC-IP) experiments confirm that hsa-miR-206-3p can bind to hsa_circ_0008726. The expressions of endogenous hsa-miR-206-3p, hsa-miR-206-3p-regulated KLF4, and KLF4-activated M2 macrophage-associated markers and chemokines were up-/down-regulated by transfection of hsa_circ_0008726 siRNAs or hsa_circ_0008726 overexpression plasmid in macrophages, respectively. These results suggest MDI-GSH exposure downregulates hsa-miR-206-3p via induction of endogenous hsa_circ_0008726/DNAJB6, resulting in the upregulation of hsa-miR-206-3p-mediated regulations in macrophages.

Keywords: 4,4′-methylene diphenyl diisocyanate (MDI); Krüppel-like factor 4 (KLF4); circular RNAs (circRNAs); hsa-miR-206-3p; occupational asthma (OA).

Figure 1

MDI-GSH conjugate treatment induces endogenous circRNA hsa_circ_0008726 in differentiated/enhanced THP-1 macrophages. Total RNA was isolated from the indicated MDI-GSH conjugate-treated differentiated/enhanced THP-1 macrophages by the mirVana^™ miR isolation kit, reverse transcribed, and subjected to SYBR green-based or TaqMan stem-loop miR RT-qPCR. Endogenous miR/circRNA expressions of (A) hsa-miR-206-3p, (B) hsa_circ_0000199, (C) hsa_circ_0001264, (D) hsa_circ_0001982, (E) hsa_circ_0004662, (F) hsa_circ_0007428, (G) hsa_circ_0008726, (H) hsa_circ_0056618, (I) hsa_circ_0057558, (J) hsa_circ_0058141, and (K) hsa_circ_0072088 were determined 24 h after MDI-GSH conjugate treatments (N = 3; bars, SEM). MDI: 4,4′-methylene diphenyl diisocyanate. GSH: Glutathione. (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 2

Circular RNA hsa_circ_0008726 is presented in THP-1 macrophages, and MDI-GSH conjugates upregulate endogenous hsa_circ_0008726 parental host gene transcript DNAJB6. (A) Characteristics of hsa_circ_0008726 obtained from the Circular RNA Interactome database. (B) Illustration shows exon numbers and designed convergent and divergent primer sites on the mature DNAJB6 transcripts. RNAse R degrades linear RNA species, including the DNAJB6 transcript. CircRNA hsa_circ_0008726 is back spliced from exon 3–5 of the DNAJB6 transcript. (C) Total RNA was isolated from THP-1 macrophages by the mirVana^™ miR isolation kit and treated with or without RNAse R, further purified using the mirVana^™ miR isolation kit, reverse transcribed, and subjected to RT-PCR using convergent or divergent primers. RT-NTC: Templates from a cDNA synthesis reaction without adding reverse transcriptase. PCR-NTC: Use only water to replace cDNA templates during PCR reaction. (D) Total RNA was isolated from MDI-GSH-treated differentiated/enhanced THP-1 macrophages at indicated concentrations for 24 h by the mirVana^™ miR isolation kit, reverse transcribed, and subjected to TaqMan RT-qPCR assays. Endogenous levels of DNAJB6 were determined at 24 h after MDI-GSH conjugate treatment (N = 3; bars, SEM). MDI: 4,4′-methylene diphenyl diisocyanate. GSH: Glutathione (** p < 0.01, *** p < 0.001).

Figure 3

Human circular RNA hsa_circ_0008726 is a target of hsa-miR-206-3p. (A) Alignment of the hsa_circ_0008726 sequence regions of potential hsa-miR-206-3p binding sites. (B) Differentiated/enhanced THP-1 macrophages were transfected with 25 nM of indicated miR-mimic or nontargeting miR-mimic control (miR-mimic-Ctl) for 24 h. The cells were collected and immunoprecipitated using the panAGO or isotype IgG antibody after 24 h transfection. RNA was isolated, and the fold enrichment of hsa_circ_0008726 was measured (N = 3; bars, SEM). (C,D) THP-1 macrophages were transfected with 25 nM of either miR-mimic/inhibitor-206-3p, miR-mimic/inhibitor-381-3p, or nontargeting miR-mimic/inhibitor control for 24 h. Total RNA was isolated from the indicated miR-mimics/inhibitors transfected THP-1 macrophages by the mirVana^™ miR isolation kit, reverse transcribed, and subjected to RT-qPCR. The endogenous hsa_circ_0008726 levels from indicated (C) miR-mimics or (D) miR-inhibitors transfected THP-1 macrophages were determined by SYBR Green RT-qPCR assays (N = 3; bars, SEM). (* p < 0.05, ** p < 0.01).

Figure 4

Transfection of hsa_circ_0008726 siRNA knocks down endogenous hsa_circ_0008726 levels and upregulates hsa-miR-206-3p in differentiated/enhanced THP-1 macrophages. Differentiated/enhanced THP-1 macrophages were transfected with 25 nM of either si-hsa_circ_0008726#1, si-hsa_circ_0008726#2 siRNA, or nontargeting siRNA control (siCtl). After 24 h, the endogenous levels of (A) hsa_circ_0008726 and (B) hsa-miR-206-3p were measured by RT-qPCR (N = 3; bars, SEM). (* p < 0.05, ** p < 0.01).

Figure 5

CircRNA hsa_circ_0008726 as a downstream effector to MDI-GSH conjugate exposure for regulating hsa-miR-206-3p/KLF4 and KLF4-mediated M2 macrophage-associated markers and chemokines in macrophages. Differentiated/enhanced THP-1 macrophages were transfected with 25 nM of either si-hsa_circ_0008726#1 or nontargeting siRNA control (siCtl) for 24 h, followed by treatment either with or without 10 µM MDI-GSH conjugate for 24 h. Total RNA was isolated from macrophages with indicated treatments/transfections by the mirVana^™ miR isolation kit, reverse transcribed, and subjected to SYBR green-based or TaqMan stem-loop miR RT-qPCR. The endogenous levels of (A) hsa_circ_0008726 and (B) hsa-miR-206-3p as well as the M2 macrophage-associated transcription factor (C) KLF4, markers (D) CD206, (E) TGM2, (F) CCL17, (G) CCL22, and (H) CCL24 mRNA levels were determined in total RNA isolated from macrophages as indicated treatments (N = 3; bars, SEM). (* p < 0.05, ** p < 0.01, *** p < 0.001 when compared to vehicle-treated macrophages with transfection of or nontargeting siRNA control (siCtl); ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, when compared to macrophages treated with 10 µM MDI-GSH conjugate as well as with transfection of indicated either si-hsa_circ_0008726#1 or nontargeting siRNA control (siCtl)).

Figure 6

Circular RNA hsa_circ_0008726 overexpression increases M2 macrophage associate markers and chemokines in differentiated/enhanced THP-1 macrophages. Differentiated/enhanced THP-1 macrophages were transfected with 2.5 µg of either pcDNA3.1⁽⁺⁾_Circ_Mini-hsa_circ_0008726 or pcDNA3.1⁽⁺⁾_Circ_Mini vector plasmids for 48 h. Total RNA was isolated from plasmids transfected THP-1 macrophages by the mirVana^™ miR isolation kit, reverse transcribed, and subjected to SYBR green or TaqMan RT-qPCR. The transgene of (A) hsa_circ_0008726 and (B) hsa-miR-206-3p as well as the endogenous M2 macrophage-associated markers (C) KLF4, (D) CD206, (E) TGM2, (F) CCL17, (G) CCL22, and (H) CCL24 mRNA levels were determined by RT-qPCR (N = 3; bars, SEM). (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 7

CircRNA hsa_circ_0008726 plays an important role for the secretion of chemokines CCL17, CCL22, and CCL24 and regulates T-cell and eosinophil chemotaxis/migration in macrophages. Cell-free conditioned media were obtained from THP-1 macrophages transfected with either the hsa_circ_0008726 overexpression plasmid or the empty vector for 48 h. The secreted protein levels of (A) CCL17, (B) CCL22, and (C) CCL24 in conditioned media from either hsa_circ_0008726 overexpressed THP-1 macrophages or empty vector transfected THP-1 macrophages were determined by ELISA according to the manufacturer’s instructions. The isolated conditioned media were used as chemoattractants to attract (D) Jurkat T-cell clone E6-1 or differentiated (E) HL-60 C_15 eosinophils. T-cell and eosinophil migration responding to the conditioned media was measured after 6 h. Percent of cells migrated towards the bottom chamber are shown (** p < 0.01, *** p < 0.001).

Figure 8

Proposed mechanisms by which MDI-GSH conjugate exposure induces M2 macrophage-associated markers and chemokine CCL17, CCL22, and CCL24 via hsa_circ_0008726/hsa-miR-206-3p-regulated KLF4 activation in macrophages. MDI: 4,4′-methylene diphenyl diisocyanate; TFs: transcription factors; CDS: coding sequences; KLF4: Krüppel-like factor 4. Note: Some illustrated schematics were obtained from motifolio templates (Motifolio Inc., Ellicott City, MD, USA).