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. 2012;7(6):e38899.
doi: 10.1371/journal.pone.0038899. Epub 2012 Jun 22.

"V体育ios版" Damage associated molecular pattern molecule-induced microRNAs (DAMPmiRs) in human peripheral blood mononuclear cells

Sebnem Unlu  1 , Siuwah Tang, Ena Wang, Ivan Martinez, Daolin Tang, Marco E Bianchi, Herbert J Zeh 3rd, Michael T Lotze
Affiliations

Damage associated molecular pattern molecule-induced microRNAs (DAMPmiRs) in human peripheral blood mononuclear cells

Sebnem Unlu et al. PLoS One. 2012.

Erratum in (V体育平台登录)

  • PLoS One. 2012;7(8). doi: 10.1371/annotation/18ec64ae-e086-446c-b63c-a71fa2bba046. Wang, E na [corrected to Wang, Ena]

VSports注册入口 - Abstract

Endogenous damage associated molecular pattern molecules (DAMPs) released from necrotic, damaged or stressed cells are associated with an inflammatory response. Whether the microRNA (miR) expression signature of this response is different from that of a pathogen associated molecular pattern (PAMP)-stimulated inflammatory response is unknown. We report here that miR-34c and miR-214 are significantly expressed in fresh human peripheral blood mononuclear cells (PBMCs) exposed to DAMP-containing freeze-thaw lysates, or to conditioned media from serum-starved and glucose-deprived cells (p<6×10(-4) and p<3. 7×10(-3)), respectively. Interestingly, only miR-34c expression was differentially expressed in PBMCs exposed to freeze-thaw lysates or conditioned media from wildtype High Mobility Group B1 (HMGB1(+/+)) mouse embryonic fibroblast (MEF) cells, when compared to cultures exposed to lysates or conditioned media from HMGB1(-/-) MEFs. miR-155 expression in these cultures was negligible, but was significantly expressed in PBMCs stimulated with Lipopolysaccahride (LPS) or most other Toll-like receptor (TLR) ligands, making it the prototypic "PAMPmiR" VSports手机版. Exposure to a damaged human colorectal carcinoma cell line lysate (HCT116) similarly resulted in increased miR-34c and miR-214 levels. When PBMCs were pre-transfected with anti-miR-34c and then exposed to lysate, expression levels of IKKγ mRNA, a putative target of miR-34c, increased, while protein levels of IKKγ in cultures transfected with a pre-miR-34c were abrogated. Levels of miR-34c expression (as well as pro-inflammatory cytokines, IL-1β and TNFα) decreased when PBMC cultures were briefly pre-incubated with the K(+) channel (inflammasome) inhibitor, glybenclamide, suggesting that inflammasome activation is upstream of miR-34c expression in response to DAMPs. Our findings demonstrate that a specific microRNA expression signature is associated with the inflammatory response to damaged/injured cells and carries implications for many acute and chronic inflammatory disorders. .

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Expression of hsa-miR-34c and hsa-miR-214 is a hallmark of human PBMCs exposed to necrotic cell lysates.
A: Hierarchical clustering or heat map of microRNA expression signatures (after real-time TaqMan RT-PCR array profiling) in donor PBMCs exposed to cell lysates and/or LPS. Total RNA extracted from PBMC cultures was run on microRNA TaqMan low-density arrays (TLDAs). B: Figure depicting changes in fold expression as log 2-transformed RQ (relative quantity) values of the statistically significant miRs (p values shown in Table 1) from each of the four donors, after exposure to the respective conditions. All values were calculated from 2−δδCt (RQ values) where the endogenous control was snoRNA U48. C: Differential expression of hsa-miR-34a, miR-34b, miR-34c and other miRs when donor PBMCs are exposed to HMGB1+/+ or HMGB1−/− lysates for 8 hrs. Data shown are average±s.d., of two independent experiments, each from a different donor, measured in triplicates, where ** indicates p<0.01, by paired Student’s t test.
Figure 2
Figure 2. Differential expression of TNFα and hsa-miR-34c in human donor PBMCs following exposure to wild-type (wt) HCT116 or HMGB1 stable knock-down (kd) lysates.
A: HCT116 cells were stably transfected with a shHMGB1 vector in the presence of Puromycin (100 ug/ml). The clone with complete knockdown of HMGB1 (indicated by the arrow) was chosen to make necrotic lysates. B: TNFα ELISA showing differential release of TNFα in human donor PBMC cell cultures following exposure to HCT116 lysates (wild-type, wt or HMGB1 knockdown, kd) for 24 hrs. The amount of HCT116 lysates used was 104 cells/ml of human PBMC culture. Data shown are the average±s.d. of three independent experiments, each from a different donor, measured in triplicates, where **indicates p<0.01, by Student’s t test. C: Fold changes in expression (as log-2-transformed RQ values) of hsa-miR-34c and hsa-miR-214 in donor PBMCs exposed to the indicated HCT116 necrotic lysates for 48 hrs. Values were measured using TaqMan real-time RT-PCR. Data shown are the average±s.d. of three independent experiments, each from an individual donor, measured in triplicates, where **indicates p<0.01, and where ***indicates p<0.001, by paired Student’s t test.
Figure 3
Figure 3. hsa-miR-34c and hsa-miR-214 are expressed at negligible levels in human PBMCs stimulated with various PAMPS or TLR ligands.
Fold changes in expression of the indicated miRs in donor PBMCs (as log 2-transformed values) after stimulation with LPS (TLR4 ligand, 100 ng/ml), Imiquimod (TLR7 ligand, 1 ug/ml), CpG ODN 2216 (TLR 9 ligand, 1 ug/ml), Pam3CSK4 (TLR 2/1 ligand, 100 ng/ml), Flagellin (TLR 5 ligand, 100 ng/ml), poly I:C (TLR 3 ligand, 50 ug/ml), R-848 (TLR 7 and TLR 8 ligand, 1 ug/ml) respectively for 48 hrs and measured using TaqMan real-time microRNA RT-PCR. Data shown are average±s.d., of two independent experiments, each from a different donor, measured in triplicate.
Figure 4
Figure 4. Changes in IKKγ mRNA and protein expression levels in human PBMCs pre-transfected with pre-miR-34c or anti-miR-34c and exposed to HMGB1+/+ or HMGB1
−/− lysates. A: Changes in fold expression (as log 2-transformed RQ values) of IKKγ mRNA levels in human PBMCs transfected with pre-miR-34c-5p or anti-miR-34c-5p and exposed to damaged HMGB1+/+ or HMGB1−/− lysates for 8 hrs. Data shown are average±s.d., of two independent experiments and normalized to the untreated (UT) samples transfected either with control miR, pre-miR-34c or anti-miR-34c, where ***indicates p<0.001, by paired Student’s t test. B: Data shown are 48 hrs after transfection of pre- miR-34c and negative control precursor oligos into donor PBMCs. Values were measured using TaqMan real-time RT-PCR and normalized to sno RNA U48. Data shown are the average±s.d. of two independent experiments, each from a different donor, measured in triplicates, where ***indicates p<0.001, by paired Student’s t test. C: Decreased protein expression of IKKγ after transfection with pre-miR-34c (lane 3) or increased protein expression of IKKγ after transfection with anti-miR-34c (lane 2) and exposure to damaged HMGB1+/+ cell lysates for 24 hrs.
Figure 5
Figure 5. Expression levels of miR-34c and miR-214 are changed when donor PBMCs are exposed to conditioned media from dying cells.
A: Changes in miR-34c, miR-214 and miR-155 expression in PMBCs (from one donor) exposed to conditioned media from HMGB1+/+ and HMGB1−/− MEF cells. PBMCs were pre- incubated with 50 µM glybenclamide (Glyb) for 30 minutes before being exposed to conditioned media (MEF CM) for 48 hrs. Heat shock (HS) was carried out at 42°C for 2 hrs. Data shown are average±s.d. from one independent experiment in triplicate, normalized to untreated control samples and RNU48 as endogenous control. B: Changes in miR-34c, miR-214 and miR-155 expression in PBMCs from another donor exposed to conditioned media. Data shown are average±s.d. from one independent experiment in triplicate, normalized to untreated control samples and RNU48 as endogenous control, where **indicates p<0.01, *indicates p<0.05, by paired Student’s t test. The two respective groups compared in Figure 5 are for each miR expression graph, the treatment at the beginning of the straight line with the treatment at the end of the line. The control lane is the first treatment in each graph, which is indicated by the white bar.
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