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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

Dicer expression was important for hepatic TNF-α expression in mice. (A) Dicer mRNA levels in the purified hepatocytes of DicerF/F mice treated with saline (Ctrl) or LPS for 1h. Each dot represented the measurement of an animal (n=5). **P<0.01 vs Ctrl. (B) Immunoblots of total proteins isolated from the purified hepatocytes of DicerF/F mice treated with LPS for 4h. MiR-125b (C) and miR-130a (D) levels in the purified hepatocytes of DicerF/F mice treated with saline (Ctrl) or LPS for 1h. Each dot represented measurement of an animal (n=5). **P<0.01 vs Ctrl. The mRNA levels of hepatic TNF-α in DicerF/FAlbCre (E) or DicerF/FMx1Cre (F) mice treated with LPS were measured, normalized to those of hepatic actin (n=5). **P<0.01 vs DicerF/F. (G) Serum TNF-α levels of mice after LPS treatment were measured by ELISA (n=5). *P<0.05, **P<0.01 vs DicerF/F. (H) Serum TNF-α levels in liposome-treated DicerF/FAlbCre or DicerF/F mice treated with LPS for 1h (n=6). **P<0.01 vs PBS, *P<0.05 vs DicerF/F or PBS (DicerF/FAlbCre).
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Figure 5: Dicer expression was important for hepatic TNF-α expression in mice. (A) Dicer mRNA levels in the purified hepatocytes of DicerF/F mice treated with saline (Ctrl) or LPS for 1h. Each dot represented the measurement of an animal (n=5). **P<0.01 vs Ctrl. (B) Immunoblots of total proteins isolated from the purified hepatocytes of DicerF/F mice treated with LPS for 4h. MiR-125b (C) and miR-130a (D) levels in the purified hepatocytes of DicerF/F mice treated with saline (Ctrl) or LPS for 1h. Each dot represented measurement of an animal (n=5). **P<0.01 vs Ctrl. The mRNA levels of hepatic TNF-α in DicerF/FAlbCre (E) or DicerF/FMx1Cre (F) mice treated with LPS were measured, normalized to those of hepatic actin (n=5). **P<0.01 vs DicerF/F. (G) Serum TNF-α levels of mice after LPS treatment were measured by ELISA (n=5). *P<0.05, **P<0.01 vs DicerF/F. (H) Serum TNF-α levels in liposome-treated DicerF/FAlbCre or DicerF/F mice treated with LPS for 1h (n=6). **P<0.01 vs PBS, *P<0.05 vs DicerF/F or PBS (DicerF/FAlbCre).

Mentions: Since LPS induced-septic shock is mostly caused by NF-κB-dependent TNF-α production 8, we next examined the in vivo roles of hepatic DICER in a mouse LPS-induced liver septic shock model. Similar as the results obtained from cultured cells, the expression levels of TNF-α (Figure 5A and 5B), miR-125b and miR-130a (Figure 5C and 5D) in mice were all greatly increased in response to LPS treatment. To study the roles of DICER in liver in LPS-induced septic shock, we generated hepatocyte-specific Dicer-deficient mice by breeding Dicerflox/flox mice (DicerF/F) with mice expressing Cre recombinase under control by either the albumin promoter (Alb-Cre) 33 or inducible Mx1 promoter (Mx1-Cre) 34, resulting in DicerF/FAlbCre or DicerF/FMx1Cre mice. Efficient deletion of Dicer in liver was confirmed by quantitative RT-PCR in these mice (Supplementary Figure 5A-C). The DicerF/FAlbCre and DicerF/FMx1Cre mice could survive to adulthood without obvious abnormalities 35. We found that administration of LPS could markedly elevated TNF-α mRNA in the livers and TNF-α protein in sera of DicerF/FAlbCre and DicerF/FMx1Cre mice, with levels significantly higher than in the control DicerF/F mice (Figure 5E-G). Agreeing well, the mature hepatic miR-125b levels were decreased (Supplementary Figure 5D). Notably, although the serum TNF-α levels were not affected, we did observe a significant decrease in miR-125b in the livers of the DicerF/+AlbCre mice after LPS treatment (Supplementary Figure 5E and 5F). Collectively, TNF-α production in Dicer mutant mice was much higher and last longer compared with wild type mice after treated with LPS.


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)

Dicer expression was important for hepatic TNF-α expression in mice. (A) Dicer mRNA levels in the purified hepatocytes of DicerF/F mice treated with saline (Ctrl) or LPS for 1h. Each dot represented the measurement of an animal (n=5). **P<0.01 vs Ctrl. (B) Immunoblots of total proteins isolated from the purified hepatocytes of DicerF/F mice treated with LPS for 4h. MiR-125b (C) and miR-130a (D) levels in the purified hepatocytes of DicerF/F mice treated with saline (Ctrl) or LPS for 1h. Each dot represented measurement of an animal (n=5). **P<0.01 vs Ctrl. The mRNA levels of hepatic TNF-α in DicerF/FAlbCre (E) or DicerF/FMx1Cre (F) mice treated with LPS were measured, normalized to those of hepatic actin (n=5). **P<0.01 vs DicerF/F. (G) Serum TNF-α levels of mice after LPS treatment were measured by ELISA (n=5). *P<0.05, **P<0.01 vs DicerF/F. (H) Serum TNF-α levels in liposome-treated DicerF/FAlbCre or DicerF/F mice treated with LPS for 1h (n=6). **P<0.01 vs PBS, *P<0.05 vs DicerF/F or PBS (DicerF/FAlbCre).
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Figure 5: Dicer expression was important for hepatic TNF-α expression in mice. (A) Dicer mRNA levels in the purified hepatocytes of DicerF/F mice treated with saline (Ctrl) or LPS for 1h. Each dot represented the measurement of an animal (n=5). **P<0.01 vs Ctrl. (B) Immunoblots of total proteins isolated from the purified hepatocytes of DicerF/F mice treated with LPS for 4h. MiR-125b (C) and miR-130a (D) levels in the purified hepatocytes of DicerF/F mice treated with saline (Ctrl) or LPS for 1h. Each dot represented measurement of an animal (n=5). **P<0.01 vs Ctrl. The mRNA levels of hepatic TNF-α in DicerF/FAlbCre (E) or DicerF/FMx1Cre (F) mice treated with LPS were measured, normalized to those of hepatic actin (n=5). **P<0.01 vs DicerF/F. (G) Serum TNF-α levels of mice after LPS treatment were measured by ELISA (n=5). *P<0.05, **P<0.01 vs DicerF/F. (H) Serum TNF-α levels in liposome-treated DicerF/FAlbCre or DicerF/F mice treated with LPS for 1h (n=6). **P<0.01 vs PBS, *P<0.05 vs DicerF/F or PBS (DicerF/FAlbCre).
Mentions: Since LPS induced-septic shock is mostly caused by NF-κB-dependent TNF-α production 8, we next examined the in vivo roles of hepatic DICER in a mouse LPS-induced liver septic shock model. Similar as the results obtained from cultured cells, the expression levels of TNF-α (Figure 5A and 5B), miR-125b and miR-130a (Figure 5C and 5D) in mice were all greatly increased in response to LPS treatment. To study the roles of DICER in liver in LPS-induced septic shock, we generated hepatocyte-specific Dicer-deficient mice by breeding Dicerflox/flox mice (DicerF/F) with mice expressing Cre recombinase under control by either the albumin promoter (Alb-Cre) 33 or inducible Mx1 promoter (Mx1-Cre) 34, resulting in DicerF/FAlbCre or DicerF/FMx1Cre mice. Efficient deletion of Dicer in liver was confirmed by quantitative RT-PCR in these mice (Supplementary Figure 5A-C). The DicerF/FAlbCre and DicerF/FMx1Cre mice could survive to adulthood without obvious abnormalities 35. We found that administration of LPS could markedly elevated TNF-α mRNA in the livers and TNF-α protein in sera of DicerF/FAlbCre and DicerF/FMx1Cre mice, with levels significantly higher than in the control DicerF/F mice (Figure 5E-G). Agreeing well, the mature hepatic miR-125b levels were decreased (Supplementary Figure 5D). Notably, although the serum TNF-α levels were not affected, we did observe a significant decrease in miR-125b in the livers of the DicerF/+AlbCre mice after LPS treatment (Supplementary Figure 5E and 5F). Collectively, TNF-α production in Dicer mutant mice was much higher and last longer compared with wild type mice after treated with LPS.

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