miR-582-5p is upregulated in patients with active tuberculosis and inhibits apoptosis of monocytes by targeting FOXO1 - PubMed (original) (raw)
miR-582-5p is upregulated in patients with active tuberculosis and inhibits apoptosis of monocytes by targeting FOXO1
Yanhua Liu et al. PLoS One. 2013.
Abstract
Macrophage apoptosis is a host innate defense mechanism against tuberculosis (TB). In this study, we found that percentage of apoptotic cells in peripheral blood monocytes from patients with active TB was lower than that from healthy controls (p<0.001). To understand whether microRNAs can modulate apoptosis of monocytes, we investigated differentially expressed microRNAs in patients with active TB. miR-582-5p was mainly expressed in monocytes and was upregulated in patients with active TB. The apoptotic percentage of THP-1 cells transfected with miR-582-5p mimics was significantly lower than those transfected with negative control of microRNA mimics (p<0.001), suggesting that miR-582-5p could inhibit apoptosis of monocytes. To our knowledge, the role of miR-582-5p in regulating apoptosis of monocytes has not been reported so far. Systematic bioinformatics analysis indicated that FOXO1 might be a target gene for miR-582-5p and its 3'UTR contains potential binding sites for miR-582-5p. To determine whether miR-582-5p could influence FOXO1 expression, miR-582-5p mimics or negative control of microRNA mimics were transfected into THP-1 cells. RT-PCR and western blot analysis showed that the miR-582-5p could suppress both FOXO1 mRNA and protein expression. Co-transfection of miR-582-5p and FOXO1 3'UTR-luciferase reporter vector into cells demonstrated that significant decrease in luciferase activity was only found in reporter vector that contained a wild type sequence of FOXO1 3'UTR, suggesting that miR-582-5p could directly target FOXO1. In conclusion, miR-582-5p inhibited apoptosis of monocytes by down-regulating FOXO1 expression and might play an important role in regulating anti-M. tuberculosis directed immune responses.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Frequencies and apoptosis of CD14+ peripheral blood monocytes in patients with active TB and healthy controls.
(A) Representative flow cytometric plots showing gating strategy and percentage of monocytes (left panel). Monocytes were defined by high CD14 expression (gate BV). Patients with active TB had significantly elevated frequency of CD14+ monocytes compared with healthy controls. Two-tailed unpaired t-test was used for statistical analysis between groups. (B) Representative flow cytometric plots showing apoptosis of monocytes (upper panel). PBMCs from patients with active TB and healthy controls were incubated with RPMI-1640 contained 2% FBS for 24 h at 37°C, and cells were stained with fluorochrome-labeled anti-human CD14, Annexin V and PI. Cells that were positive for Annexin V were defined as apoptosis. Monocytes from patients with active TB had significantly lower percentage of apoptotic cells than that from healthy controls. Mann-Whitney test was used for statistical analysis between groups.
Figure 2. miR-582-5p expression detected by real-time RT-PCR.
(A) Relative expression of miR-582-5p in CD3−CD33+, CD3−CD33−, CD3+ cell subsets and in PBMCs from patients with active TB (n = 3). miR-582-5p was mainly expressed in CD3−CD33+ cells. (B) Patients with active TB had significantly higher expression of miR-582-5p as compared with healthy controls. The relative expression of miR-582-5p, as defined by mean -ΔCt, was normalized against U6 snRNA. Two-tailed unpaired t-test was used for statistical analysis between groups.
Figure 3. Inhibition of apoptosis in monocytes by miR-582-5p.
(A) Representative flow cytometric plots showing apoptotic percentage of THP-1 cells transfected with miR-582-5p mimics or negative control of mimics (mimics NC). (B) The apoptotic percentage of THP-1 cells transfected with miR-582-5p mimics was significantly lower than those transfected with negative control of microRNA mimics (mimics NC).
Figure 4. FOXO1 is a direct target of miR-582-5p.
(A) Alignment between the predicted miR-582-5p target site of FOXO1 3′UTR region and miR-582-5p. (B) Real time RT-PCR and western blot analysis showing FOXO1 mRNA and protein expression levels in THP-1 cells transfected with miR-582-5p mimics or mimics NC, respectively. (C) Co-transfection of miR-582-5p mimics/mimics NC and FOXO1 3′UTR-luciferase reporter vector into HEK-293T cells demonstrated that significant decrease in luciferase activity was only found in reporter vector that contained a wild type sequence (FOXO1-3′UTR-wt), not in vector that contained a mutant sequence (FOXO1-3′UTR-mut) within the miR-582-5p binding site. Values were presented as relative luciferase activity after normalization to Renilla luciferase activity. FOXO1-3′UTR-wt: pMIR-FOXO1-3′UTR-wt vector; FOXO1-3′UTR-mut: pMIR-FOXO1-3′UTR-mut vector. (D) Representative flow cytometric plots showing apoptotic ratio of THP-1 transfected with FOXO1 siRNA or negative controls of siRNA (siRNA NC) (left panel). The apoptotic percentage of THP-1 cells transfected with FOXO1 siRNA were significantly lower than those transfected with negative control siRNA (siRNA NC) (p<0.01).
References
- Glaziou P, Falzon D, Floyd K, Raviglione M (2013) Global epidemiology of tuberculosis. Semin Respir Crit Care Med 34: 3–16. -PubMed
- Lawn SD, Zumla AI (2011) Tuberculosis. Lancet 378: 57–72. -PubMed
- Philips JA, Ernst JD (2012) Tuberculosis pathogenesis and immunity. Annu Rev Pathol 7: 353–384. -PubMed
- Rohde K, Yates RM, Purdy GE, Russell DG (2007) Mycobacterium tuberculosis and the environment within the phagosome. Immunol Rev 219: 37–54. -PubMed
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