Limits...
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

MiRs were upregulated to inhibit TNF-α production induced by p65/DICER signaling. Pri-miR-125b (A), pri-miR-130a (B), miR-125b (C) and miR-130a (D) levels in Huh7 cells transfected with N.S. or DICER RNAi for 72h and stimulated with LPS were determined and normalized to actin mRNA levels. *P<0.05, **P<0.01vs 0h. (E) TNF-α mRNA levels in Huh7 cells transfected with negative control (NC), miR-125b or miR-130a AS for 72h and treated with LPS were determined and normalized to actin mRNA levels. *P<0.05, **P<0.01 vs 0h. (F) TNF-α mRNA levels in Huh7 cells transfected with DICER RNAi in addition to miNC, miR-125b or miR130a for 72h and stimulated with LPS were determined and normalized to actin mRNA levels. *P<0.05 vs 0h. Pri-let-7a-1, pri-let-7a-2 (G) and mature let-7a (H) levels in Huh7 cells stimulated with LPS were determined and normalized to actin mRNA levels.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4582149&req=5

Figure 4: MiRs were upregulated to inhibit TNF-α production induced by p65/DICER signaling. Pri-miR-125b (A), pri-miR-130a (B), miR-125b (C) and miR-130a (D) levels in Huh7 cells transfected with N.S. or DICER RNAi for 72h and stimulated with LPS were determined and normalized to actin mRNA levels. *P<0.05, **P<0.01vs 0h. (E) TNF-α mRNA levels in Huh7 cells transfected with negative control (NC), miR-125b or miR-130a AS for 72h and treated with LPS were determined and normalized to actin mRNA levels. *P<0.05, **P<0.01 vs 0h. (F) TNF-α mRNA levels in Huh7 cells transfected with DICER RNAi in addition to miNC, miR-125b or miR130a for 72h and stimulated with LPS were determined and normalized to actin mRNA levels. *P<0.05 vs 0h. Pri-let-7a-1, pri-let-7a-2 (G) and mature let-7a (H) levels in Huh7 cells stimulated with LPS were determined and normalized to actin mRNA levels.

Mentions: We next attempt to study the underlying mechanism of NF-κB/p65-DICER-miRNAs axis in TNF-α production. In response to LPS treatment, the levels of pri-miR-125b and pri-miR-130a were rapidly increased in Huh7 cells (Figures 4A and 4B). Interestingly, the up-regulation of these pri-miRNAs was further enhanced when DICER was knocked down. In contrast to the changes of the pri-miRNAs, we found that the levels of the mature miR-125b and miR-130a were decreased when DICER was knocked down (Figures 4C and 4D). These results clearly showed that DICER played an important role in conversion of the pri-miR-125b and pri-miR-130a to their mature forms, and that DICER down-regulation led to an accumulation of the immature forms accompanied by the reduction of their mature forms. To provide further evidence that DICER expression driven by NF-κB indeed affects TNF-α levels, we examined the effects of miR-125b and miR-130a on TNF-α expression. The LPS-induced TNF-α mRNA levels were greatly increased when miR-125b or miR-130a were down-regulated (Figure 4E). Consistently, expressing miR-125b and miR-130a reversed the increase in TNF-α mRNA levels caused by DICER down-regulation (Figure 4F). These results supported an explanation that increases of miR-130a and miR-125b by DICER activity in the later phase of LPS treatment provides a mechanism in controlling the overproduction of TNF-α. Since TLR-induced gene expression is generally mediated by NF-κB 31, we further examined the levels of both primary and mature forms of let-7a, a miRNA known to target IL-6 32. Neither primary nor mature form of let-7a was affected (Figure 4G and 4H), suggesting that regulation of IL-6 production was different from that of TNF-α. Taken together, the above results revealed a regulatory mechanism involving NF-κB/p65, DICER, miR-125b and miR-130a that controls TNF-α homeostatic production in response to NF-κB activation in hepatocytes.


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)

MiRs were upregulated to inhibit TNF-α production induced by p65/DICER signaling. Pri-miR-125b (A), pri-miR-130a (B), miR-125b (C) and miR-130a (D) levels in Huh7 cells transfected with N.S. or DICER RNAi for 72h and stimulated with LPS were determined and normalized to actin mRNA levels. *P<0.05, **P<0.01vs 0h. (E) TNF-α mRNA levels in Huh7 cells transfected with negative control (NC), miR-125b or miR-130a AS for 72h and treated with LPS were determined and normalized to actin mRNA levels. *P<0.05, **P<0.01 vs 0h. (F) TNF-α mRNA levels in Huh7 cells transfected with DICER RNAi in addition to miNC, miR-125b or miR130a for 72h and stimulated with LPS were determined and normalized to actin mRNA levels. *P<0.05 vs 0h. Pri-let-7a-1, pri-let-7a-2 (G) and mature let-7a (H) levels in Huh7 cells stimulated with LPS were determined and normalized to actin mRNA levels.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4582149&req=5

Figure 4: MiRs were upregulated to inhibit TNF-α production induced by p65/DICER signaling. Pri-miR-125b (A), pri-miR-130a (B), miR-125b (C) and miR-130a (D) levels in Huh7 cells transfected with N.S. or DICER RNAi for 72h and stimulated with LPS were determined and normalized to actin mRNA levels. *P<0.05, **P<0.01vs 0h. (E) TNF-α mRNA levels in Huh7 cells transfected with negative control (NC), miR-125b or miR-130a AS for 72h and treated with LPS were determined and normalized to actin mRNA levels. *P<0.05, **P<0.01 vs 0h. (F) TNF-α mRNA levels in Huh7 cells transfected with DICER RNAi in addition to miNC, miR-125b or miR130a for 72h and stimulated with LPS were determined and normalized to actin mRNA levels. *P<0.05 vs 0h. Pri-let-7a-1, pri-let-7a-2 (G) and mature let-7a (H) levels in Huh7 cells stimulated with LPS were determined and normalized to actin mRNA levels.
Mentions: We next attempt to study the underlying mechanism of NF-κB/p65-DICER-miRNAs axis in TNF-α production. In response to LPS treatment, the levels of pri-miR-125b and pri-miR-130a were rapidly increased in Huh7 cells (Figures 4A and 4B). Interestingly, the up-regulation of these pri-miRNAs was further enhanced when DICER was knocked down. In contrast to the changes of the pri-miRNAs, we found that the levels of the mature miR-125b and miR-130a were decreased when DICER was knocked down (Figures 4C and 4D). These results clearly showed that DICER played an important role in conversion of the pri-miR-125b and pri-miR-130a to their mature forms, and that DICER down-regulation led to an accumulation of the immature forms accompanied by the reduction of their mature forms. To provide further evidence that DICER expression driven by NF-κB indeed affects TNF-α levels, we examined the effects of miR-125b and miR-130a on TNF-α expression. The LPS-induced TNF-α mRNA levels were greatly increased when miR-125b or miR-130a were down-regulated (Figure 4E). Consistently, expressing miR-125b and miR-130a reversed the increase in TNF-α mRNA levels caused by DICER down-regulation (Figure 4F). These results supported an explanation that increases of miR-130a and miR-125b by DICER activity in the later phase of LPS treatment provides a mechanism in controlling the overproduction of TNF-α. Since TLR-induced gene expression is generally mediated by NF-κB 31, we further examined the levels of both primary and mature forms of let-7a, a miRNA known to target IL-6 32. Neither primary nor mature form of let-7a was affected (Figure 4G and 4H), suggesting that regulation of IL-6 production was different from that of TNF-α. Taken together, the above results revealed a regulatory mechanism involving NF-κB/p65, DICER, miR-125b and miR-130a that controls TNF-α homeostatic production in response to NF-κB activation in hepatocytes.

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