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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

MiR-125b and miR-130a expression was regulated by NF-κB/DICER pathway. (A) Top: Sequence alignment of miR-125b and its target sites in 3′-UTR of TNF-α in human. Gray Characters: the mutant seed sequence. Lower: A luciferase reporter construct containing a ~240bp fragment of the TNF-α 3′-UTR with the miR-125b binding site was made. The luciferase activity in HEK293 cells transfected with Wild type (WT) or mutant reporter plasmids (Mutant) and miR-125b (miR-125b) or empty plasmid (Ctrl) for 48h was determined. **P<0.01 vs Ctrl. (B) Top: Sequence alignment of miR-130a and its target sites in 3′-UTR of TNF-α in human. Gray Characters: the mutant seed sequence. Lower: A luciferase reporter construct containing a ~240bp fragment of the TNF-α 3′-UTR with the miR-130a binding site was made. The luciferase activity in HEK293 cells transfected with Wild type (WT) or mutant reporter plasmids (Mutant) and miR-130a (miR-130a) or empty plasmid (Ctrl) for 48h was determined. **P<0.01vs Ctrl. The expression of miR-125b or miR-130a in Huh7 cells transfected with GFP, DICER or p65-GFP (C and E) for 48h; N.S., DICER RNAi or p65-si1 (D and F) for 72h was determined by qRT-PCR. *P<0.05, **P<0.01 vs GFP or N.S. The levels of miR-125b (G) or miR-130a (I) in Huh7 cells cotransfected with N.S. and GFP (N.C.) or DICER RNAi and p65-GFP for 72h were determined. *P<0.05, **P<0.01 vs N.C. MiR-125b (H) or miR-130a (J) levels in Huh7 cells transfected with N.S. or DICER RNAi for 72h and treated with LPS for 4h were determined., **P<0.01 vs N.S. All quantitative data were means ± SEM of three independent experiments in triplicates. The levels of miR-125b and miR-130a were normalized to those of actin mRNA.
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Figure 3: MiR-125b and miR-130a expression was regulated by NF-κB/DICER pathway. (A) Top: Sequence alignment of miR-125b and its target sites in 3′-UTR of TNF-α in human. Gray Characters: the mutant seed sequence. Lower: A luciferase reporter construct containing a ~240bp fragment of the TNF-α 3′-UTR with the miR-125b binding site was made. The luciferase activity in HEK293 cells transfected with Wild type (WT) or mutant reporter plasmids (Mutant) and miR-125b (miR-125b) or empty plasmid (Ctrl) for 48h was determined. **P<0.01 vs Ctrl. (B) Top: Sequence alignment of miR-130a and its target sites in 3′-UTR of TNF-α in human. Gray Characters: the mutant seed sequence. Lower: A luciferase reporter construct containing a ~240bp fragment of the TNF-α 3′-UTR with the miR-130a binding site was made. The luciferase activity in HEK293 cells transfected with Wild type (WT) or mutant reporter plasmids (Mutant) and miR-130a (miR-130a) or empty plasmid (Ctrl) for 48h was determined. **P<0.01vs Ctrl. The expression of miR-125b or miR-130a in Huh7 cells transfected with GFP, DICER or p65-GFP (C and E) for 48h; N.S., DICER RNAi or p65-si1 (D and F) for 72h was determined by qRT-PCR. *P<0.05, **P<0.01 vs GFP or N.S. The levels of miR-125b (G) or miR-130a (I) in Huh7 cells cotransfected with N.S. and GFP (N.C.) or DICER RNAi and p65-GFP for 72h were determined. *P<0.05, **P<0.01 vs N.C. MiR-125b (H) or miR-130a (J) levels in Huh7 cells transfected with N.S. or DICER RNAi for 72h and treated with LPS for 4h were determined., **P<0.01 vs N.S. All quantitative data were means ± SEM of three independent experiments in triplicates. The levels of miR-125b and miR-130a were normalized to those of actin mRNA.

Mentions: DICER is an RNase III responsible for production of mature miRNAs. To determine which miRNAs mediate the potential inhibitory effects of DICER on TNF-α production, we analyzed the 3'-UTR of TNF-α mRNA and found that, miR-125b, miR-19, miR-181, miR-130a and miR-16 target sequences which were reported previously affecting TNF-α expression 16, 27-30 (Supplementary Figure 4A). Through expressing these miRNAs in HEK293 cells, we found that miR-125b and miR-130a could decrease both TNF-α mRNA levels (Supplementary Figure 4B) and the luciferase activities of a luciferase construct with the TNF-α 3'-UTR (Figure 3A and 3B). Furthermore, through overexpression or RNAi knockdown of either p65 or DICER, we did find that the expression of both miR-125b and miR-130a was regulated by p65 as well as DICER (Figure 3C-3F). Knockdown experiments further verified the requirement of DICER for either LPS-induced and p65-dependent expression of both miR-125b and miR-130a (Figure 3G-3J). These results suggest that up-regulation of miR-125b and miR-130a by the NF-κB/p65/DICER pathway may be an important mechanism preventing overproduction of TNF-α.


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)

MiR-125b and miR-130a expression was regulated by NF-κB/DICER pathway. (A) Top: Sequence alignment of miR-125b and its target sites in 3′-UTR of TNF-α in human. Gray Characters: the mutant seed sequence. Lower: A luciferase reporter construct containing a ~240bp fragment of the TNF-α 3′-UTR with the miR-125b binding site was made. The luciferase activity in HEK293 cells transfected with Wild type (WT) or mutant reporter plasmids (Mutant) and miR-125b (miR-125b) or empty plasmid (Ctrl) for 48h was determined. **P<0.01 vs Ctrl. (B) Top: Sequence alignment of miR-130a and its target sites in 3′-UTR of TNF-α in human. Gray Characters: the mutant seed sequence. Lower: A luciferase reporter construct containing a ~240bp fragment of the TNF-α 3′-UTR with the miR-130a binding site was made. The luciferase activity in HEK293 cells transfected with Wild type (WT) or mutant reporter plasmids (Mutant) and miR-130a (miR-130a) or empty plasmid (Ctrl) for 48h was determined. **P<0.01vs Ctrl. The expression of miR-125b or miR-130a in Huh7 cells transfected with GFP, DICER or p65-GFP (C and E) for 48h; N.S., DICER RNAi or p65-si1 (D and F) for 72h was determined by qRT-PCR. *P<0.05, **P<0.01 vs GFP or N.S. The levels of miR-125b (G) or miR-130a (I) in Huh7 cells cotransfected with N.S. and GFP (N.C.) or DICER RNAi and p65-GFP for 72h were determined. *P<0.05, **P<0.01 vs N.C. MiR-125b (H) or miR-130a (J) levels in Huh7 cells transfected with N.S. or DICER RNAi for 72h and treated with LPS for 4h were determined., **P<0.01 vs N.S. All quantitative data were means ± SEM of three independent experiments in triplicates. The levels of miR-125b and miR-130a were normalized to those of actin mRNA.
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Figure 3: MiR-125b and miR-130a expression was regulated by NF-κB/DICER pathway. (A) Top: Sequence alignment of miR-125b and its target sites in 3′-UTR of TNF-α in human. Gray Characters: the mutant seed sequence. Lower: A luciferase reporter construct containing a ~240bp fragment of the TNF-α 3′-UTR with the miR-125b binding site was made. The luciferase activity in HEK293 cells transfected with Wild type (WT) or mutant reporter plasmids (Mutant) and miR-125b (miR-125b) or empty plasmid (Ctrl) for 48h was determined. **P<0.01 vs Ctrl. (B) Top: Sequence alignment of miR-130a and its target sites in 3′-UTR of TNF-α in human. Gray Characters: the mutant seed sequence. Lower: A luciferase reporter construct containing a ~240bp fragment of the TNF-α 3′-UTR with the miR-130a binding site was made. The luciferase activity in HEK293 cells transfected with Wild type (WT) or mutant reporter plasmids (Mutant) and miR-130a (miR-130a) or empty plasmid (Ctrl) for 48h was determined. **P<0.01vs Ctrl. The expression of miR-125b or miR-130a in Huh7 cells transfected with GFP, DICER or p65-GFP (C and E) for 48h; N.S., DICER RNAi or p65-si1 (D and F) for 72h was determined by qRT-PCR. *P<0.05, **P<0.01 vs GFP or N.S. The levels of miR-125b (G) or miR-130a (I) in Huh7 cells cotransfected with N.S. and GFP (N.C.) or DICER RNAi and p65-GFP for 72h were determined. *P<0.05, **P<0.01 vs N.C. MiR-125b (H) or miR-130a (J) levels in Huh7 cells transfected with N.S. or DICER RNAi for 72h and treated with LPS for 4h were determined., **P<0.01 vs N.S. All quantitative data were means ± SEM of three independent experiments in triplicates. The levels of miR-125b and miR-130a were normalized to those of actin mRNA.
Mentions: DICER is an RNase III responsible for production of mature miRNAs. To determine which miRNAs mediate the potential inhibitory effects of DICER on TNF-α production, we analyzed the 3'-UTR of TNF-α mRNA and found that, miR-125b, miR-19, miR-181, miR-130a and miR-16 target sequences which were reported previously affecting TNF-α expression 16, 27-30 (Supplementary Figure 4A). Through expressing these miRNAs in HEK293 cells, we found that miR-125b and miR-130a could decrease both TNF-α mRNA levels (Supplementary Figure 4B) and the luciferase activities of a luciferase construct with the TNF-α 3'-UTR (Figure 3A and 3B). Furthermore, through overexpression or RNAi knockdown of either p65 or DICER, we did find that the expression of both miR-125b and miR-130a was regulated by p65 as well as DICER (Figure 3C-3F). Knockdown experiments further verified the requirement of DICER for either LPS-induced and p65-dependent expression of both miR-125b and miR-130a (Figure 3G-3J). These results suggest that up-regulation of miR-125b and miR-130a by the NF-κB/p65/DICER pathway may be an important mechanism preventing overproduction of TNF-α.

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