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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-induced DICER expression suppressed TNF-α production. (A) Huh7 cells treated with LPS for the indicated times were harvested for immunoblot. (B) Huh7 cells transfected with Scr or p65-si1 for 72h and treated with LPS for 4h were lysated for immunoblot. (C) The mRNA levels of DICER and TNF-α in Huh7 cells stimulated by LPS and normalized to those of actin. *P<0.05, **P<0.01, ***P<0.001 vs 0h. The mRNA levels of TNF-α (D), IL-6 (E) and IL-8 (F) in Huh7 cells transfected with N.S. or DICER RNAi plasmids (DICER RNAi) for 72h and treated with LPS and normalized to those of actin. #P<0.05, ##P<0.01 vs N.S..
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Figure 2: NF-κB /p65-induced DICER expression suppressed TNF-α production. (A) Huh7 cells treated with LPS for the indicated times were harvested for immunoblot. (B) Huh7 cells transfected with Scr or p65-si1 for 72h and treated with LPS for 4h were lysated for immunoblot. (C) The mRNA levels of DICER and TNF-α in Huh7 cells stimulated by LPS and normalized to those of actin. *P<0.05, **P<0.01, ***P<0.001 vs 0h. The mRNA levels of TNF-α (D), IL-6 (E) and IL-8 (F) in Huh7 cells transfected with N.S. or DICER RNAi plasmids (DICER RNAi) for 72h and treated with LPS and normalized to those of actin. #P<0.05, ##P<0.01 vs N.S..

Mentions: To explore the functional regulation of NF-κB/p65-DICER axis, we treated Huh7 cells with LPS and followed DICER and TNF-α expression. DICER protein levels in Huh7 cells were increased between 1h and 4h after LPS treatment (Figure 2A). Interestingly, its levels in RAW 264.7 cells, a mouse macrophage cell line known abundant for TNF-α production 24, were not changed (Supplementary Figure 3A and 3B). LPS did not increase DICER expression when p65 was down-regulated (Figure 2B). In the time course analysis of expression of DICER and TNF-α, we found that DICER mRNA levels were enhanced 1h with the peak at 4h, while TNF-α was at 2h with the peak level at 3h after LPS treatment (Figure 2C). This LPS-increased TNF-α mRNA level was greatly enhanced with the peak level at 4h when DICER was down-regulated by DICER RNAi (Figure 2D), while NF-κB/p65 activation remained unaffected (Supplementary Figure 3C and 3D). In contrast, the mRNA levels of IL-6 (Figure 2E) and IL-8 (Figure 2F), two inflammatory cytokines also regulated by NF-κB 25, 26, remained unaffected by DICER knockdown in response to LPS. These results suggested that up-regulation of DICER following NF-κB/p65 activation may play an important role in preventing overproduction of TNF-α in Huh7 cells.


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-induced DICER expression suppressed TNF-α production. (A) Huh7 cells treated with LPS for the indicated times were harvested for immunoblot. (B) Huh7 cells transfected with Scr or p65-si1 for 72h and treated with LPS for 4h were lysated for immunoblot. (C) The mRNA levels of DICER and TNF-α in Huh7 cells stimulated by LPS and normalized to those of actin. *P<0.05, **P<0.01, ***P<0.001 vs 0h. The mRNA levels of TNF-α (D), IL-6 (E) and IL-8 (F) in Huh7 cells transfected with N.S. or DICER RNAi plasmids (DICER RNAi) for 72h and treated with LPS and normalized to those of actin. #P<0.05, ##P<0.01 vs N.S..
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getmorefigures.php?uid=PMC4582149&req=5

Figure 2: NF-κB /p65-induced DICER expression suppressed TNF-α production. (A) Huh7 cells treated with LPS for the indicated times were harvested for immunoblot. (B) Huh7 cells transfected with Scr or p65-si1 for 72h and treated with LPS for 4h were lysated for immunoblot. (C) The mRNA levels of DICER and TNF-α in Huh7 cells stimulated by LPS and normalized to those of actin. *P<0.05, **P<0.01, ***P<0.001 vs 0h. The mRNA levels of TNF-α (D), IL-6 (E) and IL-8 (F) in Huh7 cells transfected with N.S. or DICER RNAi plasmids (DICER RNAi) for 72h and treated with LPS and normalized to those of actin. #P<0.05, ##P<0.01 vs N.S..
Mentions: To explore the functional regulation of NF-κB/p65-DICER axis, we treated Huh7 cells with LPS and followed DICER and TNF-α expression. DICER protein levels in Huh7 cells were increased between 1h and 4h after LPS treatment (Figure 2A). Interestingly, its levels in RAW 264.7 cells, a mouse macrophage cell line known abundant for TNF-α production 24, were not changed (Supplementary Figure 3A and 3B). LPS did not increase DICER expression when p65 was down-regulated (Figure 2B). In the time course analysis of expression of DICER and TNF-α, we found that DICER mRNA levels were enhanced 1h with the peak at 4h, while TNF-α was at 2h with the peak level at 3h after LPS treatment (Figure 2C). This LPS-increased TNF-α mRNA level was greatly enhanced with the peak level at 4h when DICER was down-regulated by DICER RNAi (Figure 2D), while NF-κB/p65 activation remained unaffected (Supplementary Figure 3C and 3D). In contrast, the mRNA levels of IL-6 (Figure 2E) and IL-8 (Figure 2F), two inflammatory cytokines also regulated by NF-κB 25, 26, remained unaffected by DICER knockdown in response to LPS. These results suggested that up-regulation of DICER following NF-κB/p65 activation may play an important role in preventing overproduction of TNF-α in Huh7 cells.

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