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NF-κB-DICER-miRs Axis Regulates TNF-α Expression in Responses to Endotoxin Stress.

Guan Y, Yao H, Wang J, Sun K, Cao L, Wang Y - Int. J. Biol. Sci. (2015)

Bottom Line: Unbalanced tumor necrosis factor (TNF)-α production is associated with pathogenesis of a variety of human diseases.We demonstrated that NF-κB bound to DICER promoter and transcriptionally regulated DICER expression.Furthermore, we showed that the hepatocyte-specific depletion of Dicer in mice resulted in TNF-α overproduction and sensitized the mice to endotoxin, which could be corrected by administration of miR-125b mimics.

View Article: PubMed Central - PubMed

Affiliation: 1. Key Laboratory of Medical Cell Biology, China Medical University, Shenyang, 110122, China.

ABSTRACT
Unbalanced tumor necrosis factor (TNF)-α production is associated with pathogenesis of a variety of human diseases. However, the molecular pathways maintaining TNF-α homeostasis remain elusive. Here, we report that NF-κB/p65-DICER-miRs axis negatively regulates TNF-α production. We demonstrated that NF-κB bound to DICER promoter and transcriptionally regulated DICER expression. In addition, the NF-κB/DICER signaling suppresses TNF-α expression by generating mature forms of miR-125b and miR-130a which negatively regulate TNF-α mRNA. Furthermore, we showed that the hepatocyte-specific depletion of Dicer in mice resulted in TNF-α overproduction and sensitized the mice to endotoxin, which could be corrected by administration of miR-125b mimics. These data suggest that NF-κB/p65-DICER-miRs axis involved in maintaining of TNF-α homeostasis, and injection of miR-125b as a potential therapeutic method for septic shock.

No MeSH data available.


Related in: MedlinePlus

NF-κB /p65 regulation of DICER expression. (A) DICER promoter analysis. HEK293 and HeLa cells were transiently transfected with firefly luciferase reporter constructs (pGL3-basic) containing the indicated genomic fragments (Left, schematic diagram) upstream of DICER and analyzed for luciferase activities. All luciferase data were normalized to β-galactosidase. Fold changes were shown with respect to pGL3-basic, where the normalized activity was set to value of 1. (B) VISTA schematic diagram showed the phylogenetic conservation between human, mouse and rat in the genomic region upstream of DICER. The sequences of predicted κB site (DICER κB probe) and mutated κB (mκB) site. (C) Huh7 cells were transfected with the proximal promoter construct pGL3 (-450/-180) (C) or mκB-pGL3 (-450/-180) (MUT) and subsequently treated with LPS for 4h, TNF-α for 2h, APDC for 8h, BMS or BAY for 1h, and luciferase activity was determined. *P<0.05, **P<0.01, ***P<0.001 vs C. (D) Luciferase activities in HEK293 cells transfected with pGL3 (-450/-180) and nonsense siRNA (N.S.), p65-specific siRNA (p65-si1), p65-GFP or empty vector (GFP) each. The activities were relative to those in N.S. or GFP groups. Immunoblots (inset) showing the knockdown or overexpression of p65. *P<0.05, **P<0.01 vs N.S. or GFP. (E) NF-κB/p65 interaction with DICER promoter analyzed by ChIP assay. EB staining, qPCR products on a 2% agarose gel; bar graph, statistical analysis of PCR. Data were normalized with the input DNA. **P<0.01 vs Saline. Wild-type (WT) or p65 KO (p65-/-) MEFs were treated with LPS or TNF-α for the indicated times and harvested for qRT-PCR (F and H) or immunoblot (G and I). The mRNA levels of DICER were normalized to those of actin and relative to those in non-stimulated cells. *P<0.05, **P<0.01 vs 0h. #P<0.05, ##P<0.01, ###P<0.001 vs WT. All quantitative data, unless stated, were means ± SEM of three independent experiments in triplicates and tubulin served as a loading control in all immunoblots.
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Figure 1: NF-κB /p65 regulation of DICER expression. (A) DICER promoter analysis. HEK293 and HeLa cells were transiently transfected with firefly luciferase reporter constructs (pGL3-basic) containing the indicated genomic fragments (Left, schematic diagram) upstream of DICER and analyzed for luciferase activities. All luciferase data were normalized to β-galactosidase. Fold changes were shown with respect to pGL3-basic, where the normalized activity was set to value of 1. (B) VISTA schematic diagram showed the phylogenetic conservation between human, mouse and rat in the genomic region upstream of DICER. The sequences of predicted κB site (DICER κB probe) and mutated κB (mκB) site. (C) Huh7 cells were transfected with the proximal promoter construct pGL3 (-450/-180) (C) or mκB-pGL3 (-450/-180) (MUT) and subsequently treated with LPS for 4h, TNF-α for 2h, APDC for 8h, BMS or BAY for 1h, and luciferase activity was determined. *P<0.05, **P<0.01, ***P<0.001 vs C. (D) Luciferase activities in HEK293 cells transfected with pGL3 (-450/-180) and nonsense siRNA (N.S.), p65-specific siRNA (p65-si1), p65-GFP or empty vector (GFP) each. The activities were relative to those in N.S. or GFP groups. Immunoblots (inset) showing the knockdown or overexpression of p65. *P<0.05, **P<0.01 vs N.S. or GFP. (E) NF-κB/p65 interaction with DICER promoter analyzed by ChIP assay. EB staining, qPCR products on a 2% agarose gel; bar graph, statistical analysis of PCR. Data were normalized with the input DNA. **P<0.01 vs Saline. Wild-type (WT) or p65 KO (p65-/-) MEFs were treated with LPS or TNF-α for the indicated times and harvested for qRT-PCR (F and H) or immunoblot (G and I). The mRNA levels of DICER were normalized to those of actin and relative to those in non-stimulated cells. *P<0.05, **P<0.01 vs 0h. #P<0.05, ##P<0.01, ###P<0.001 vs WT. All quantitative data, unless stated, were means ± SEM of three independent experiments in triplicates and tubulin served as a loading control in all immunoblots.

Mentions: To analysis the transcriptional regulation of DICER, we first mapped the regions of DICER promoter and found that its core promoter is located at -450 to -180 bp relative to its transcription start site (Figure 1A, 1B and Supplementary Figure 1A), a region highly conserved among human, mouse and rat. Further the TRANSFAC analysis revealed the presence of an NF-κB binding site in this region (Figure 1B). Mutagenic analysis confirmed this putative NF-κB regulatory element in DICER promoter was functional in both Huh7 cells, a human hepatocarcinoma cell line (Figure 1C), and HEK293 cells (Supplementary Figure 1B). Further, siRNA knockdown and overexpression experiments showed that the p65 subunit rather than the p50 subunit was important for DICER promoter activity (Figure 1D, Supplementary Figure 1C and 1D, and Supplemental Figure 2). Additionally, electrophoretic mobility shift assays (EMSA) (Supplementary Figure 1E and 1F) and chromatin immunoprecipitation (ChIP) assays (Figure 1E and Supplementary Figure 1G) revealed that DICER promoter was physically associated with NF-κB/p65. Consistently, DICER expression induced by LPS (Figure 1F and 1G), a bacterial endotoxin responsible for septic shock 23, and by TNF-α (Figure 1H and 1I) in mouse embryonic fibroblasts (MEF) deficient for p65 was prevented. Taken together, these results demonstrate that NF-κB/p65 regulates DICER expression.


NF-κB-DICER-miRs Axis Regulates TNF-α Expression in Responses to Endotoxin Stress.

Guan Y, Yao H, Wang J, Sun K, Cao L, Wang Y - Int. J. Biol. Sci. (2015)

NF-κB /p65 regulation of DICER expression. (A) DICER promoter analysis. HEK293 and HeLa cells were transiently transfected with firefly luciferase reporter constructs (pGL3-basic) containing the indicated genomic fragments (Left, schematic diagram) upstream of DICER and analyzed for luciferase activities. All luciferase data were normalized to β-galactosidase. Fold changes were shown with respect to pGL3-basic, where the normalized activity was set to value of 1. (B) VISTA schematic diagram showed the phylogenetic conservation between human, mouse and rat in the genomic region upstream of DICER. The sequences of predicted κB site (DICER κB probe) and mutated κB (mκB) site. (C) Huh7 cells were transfected with the proximal promoter construct pGL3 (-450/-180) (C) or mκB-pGL3 (-450/-180) (MUT) and subsequently treated with LPS for 4h, TNF-α for 2h, APDC for 8h, BMS or BAY for 1h, and luciferase activity was determined. *P<0.05, **P<0.01, ***P<0.001 vs C. (D) Luciferase activities in HEK293 cells transfected with pGL3 (-450/-180) and nonsense siRNA (N.S.), p65-specific siRNA (p65-si1), p65-GFP or empty vector (GFP) each. The activities were relative to those in N.S. or GFP groups. Immunoblots (inset) showing the knockdown or overexpression of p65. *P<0.05, **P<0.01 vs N.S. or GFP. (E) NF-κB/p65 interaction with DICER promoter analyzed by ChIP assay. EB staining, qPCR products on a 2% agarose gel; bar graph, statistical analysis of PCR. Data were normalized with the input DNA. **P<0.01 vs Saline. Wild-type (WT) or p65 KO (p65-/-) MEFs were treated with LPS or TNF-α for the indicated times and harvested for qRT-PCR (F and H) or immunoblot (G and I). The mRNA levels of DICER were normalized to those of actin and relative to those in non-stimulated cells. *P<0.05, **P<0.01 vs 0h. #P<0.05, ##P<0.01, ###P<0.001 vs WT. All quantitative data, unless stated, were means ± SEM of three independent experiments in triplicates and tubulin served as a loading control in all immunoblots.
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Related In: Results  -  Collection

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Figure 1: NF-κB /p65 regulation of DICER expression. (A) DICER promoter analysis. HEK293 and HeLa cells were transiently transfected with firefly luciferase reporter constructs (pGL3-basic) containing the indicated genomic fragments (Left, schematic diagram) upstream of DICER and analyzed for luciferase activities. All luciferase data were normalized to β-galactosidase. Fold changes were shown with respect to pGL3-basic, where the normalized activity was set to value of 1. (B) VISTA schematic diagram showed the phylogenetic conservation between human, mouse and rat in the genomic region upstream of DICER. The sequences of predicted κB site (DICER κB probe) and mutated κB (mκB) site. (C) Huh7 cells were transfected with the proximal promoter construct pGL3 (-450/-180) (C) or mκB-pGL3 (-450/-180) (MUT) and subsequently treated with LPS for 4h, TNF-α for 2h, APDC for 8h, BMS or BAY for 1h, and luciferase activity was determined. *P<0.05, **P<0.01, ***P<0.001 vs C. (D) Luciferase activities in HEK293 cells transfected with pGL3 (-450/-180) and nonsense siRNA (N.S.), p65-specific siRNA (p65-si1), p65-GFP or empty vector (GFP) each. The activities were relative to those in N.S. or GFP groups. Immunoblots (inset) showing the knockdown or overexpression of p65. *P<0.05, **P<0.01 vs N.S. or GFP. (E) NF-κB/p65 interaction with DICER promoter analyzed by ChIP assay. EB staining, qPCR products on a 2% agarose gel; bar graph, statistical analysis of PCR. Data were normalized with the input DNA. **P<0.01 vs Saline. Wild-type (WT) or p65 KO (p65-/-) MEFs were treated with LPS or TNF-α for the indicated times and harvested for qRT-PCR (F and H) or immunoblot (G and I). The mRNA levels of DICER were normalized to those of actin and relative to those in non-stimulated cells. *P<0.05, **P<0.01 vs 0h. #P<0.05, ##P<0.01, ###P<0.001 vs WT. All quantitative data, unless stated, were means ± SEM of three independent experiments in triplicates and tubulin served as a loading control in all immunoblots.
Mentions: To analysis the transcriptional regulation of DICER, we first mapped the regions of DICER promoter and found that its core promoter is located at -450 to -180 bp relative to its transcription start site (Figure 1A, 1B and Supplementary Figure 1A), a region highly conserved among human, mouse and rat. Further the TRANSFAC analysis revealed the presence of an NF-κB binding site in this region (Figure 1B). Mutagenic analysis confirmed this putative NF-κB regulatory element in DICER promoter was functional in both Huh7 cells, a human hepatocarcinoma cell line (Figure 1C), and HEK293 cells (Supplementary Figure 1B). Further, siRNA knockdown and overexpression experiments showed that the p65 subunit rather than the p50 subunit was important for DICER promoter activity (Figure 1D, Supplementary Figure 1C and 1D, and Supplemental Figure 2). Additionally, electrophoretic mobility shift assays (EMSA) (Supplementary Figure 1E and 1F) and chromatin immunoprecipitation (ChIP) assays (Figure 1E and Supplementary Figure 1G) revealed that DICER promoter was physically associated with NF-κB/p65. Consistently, DICER expression induced by LPS (Figure 1F and 1G), a bacterial endotoxin responsible for septic shock 23, and by TNF-α (Figure 1H and 1I) in mouse embryonic fibroblasts (MEF) deficient for p65 was prevented. Taken together, these results demonstrate that NF-κB/p65 regulates DICER expression.

Bottom Line: Unbalanced tumor necrosis factor (TNF)-α production is associated with pathogenesis of a variety of human diseases.We demonstrated that NF-κB bound to DICER promoter and transcriptionally regulated DICER expression.Furthermore, we showed that the hepatocyte-specific depletion of Dicer in mice resulted in TNF-α overproduction and sensitized the mice to endotoxin, which could be corrected by administration of miR-125b mimics.

View Article: PubMed Central - PubMed

Affiliation: 1. Key Laboratory of Medical Cell Biology, China Medical University, Shenyang, 110122, China.

ABSTRACT
Unbalanced tumor necrosis factor (TNF)-α production is associated with pathogenesis of a variety of human diseases. However, the molecular pathways maintaining TNF-α homeostasis remain elusive. Here, we report that NF-κB/p65-DICER-miRs axis negatively regulates TNF-α production. We demonstrated that NF-κB bound to DICER promoter and transcriptionally regulated DICER expression. In addition, the NF-κB/DICER signaling suppresses TNF-α expression by generating mature forms of miR-125b and miR-130a which negatively regulate TNF-α mRNA. Furthermore, we showed that the hepatocyte-specific depletion of Dicer in mice resulted in TNF-α overproduction and sensitized the mice to endotoxin, which could be corrected by administration of miR-125b mimics. These data suggest that NF-κB/p65-DICER-miRs axis involved in maintaining of TNF-α homeostasis, and injection of miR-125b as a potential therapeutic method for septic shock.

No MeSH data available.


Related in: MedlinePlus