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Temporal regulation of HIF-1 and NF-κB in hypoxic hepatocarcinoma cells.

Jiang Y, Zhu Y, Wang X, Gong J, Hu C, Guo B, Zhu B, Li Y - Oncotarget (2015)

Bottom Line: Regulations between NF-κB and HIF-1 have not been adequately addressed in previous research.We also found that NF-κB p50 and p65 (RelA), but not c-Rel, bound the HIF-1a promoter, thus increasing its transcription.Dicer1, a key enzyme in miRNA biogenesis, was decreased by acute hypoxia but was later increased by HIF-1, rather than by the above-mentioned NF-κB subunits.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cancer, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China.

ABSTRACT
Regulations between NF-κB and HIF-1 have not been adequately addressed in previous research. Here, we report that hypoxia increased NF-κB in hepatocellular carcinoma cells. The HIF-1 protein level was rapidly induced by protein stabilization (by 2 hours) and then moderately decreased, whereas mRNA levels were reciprocally increased. We also found that NF-κB p50 and p65 (RelA), but not c-Rel, bound the HIF-1a promoter, thus increasing its transcription. In contrast, miR-199a-5p and miR-93, c-Rel downstream targets, decreased HIF-1α at both the mRNA and protein levels. Dicer1, a key enzyme in miRNA biogenesis, was decreased by acute hypoxia but was later increased by HIF-1, rather than by the above-mentioned NF-κB subunits. Thus, NF-κB both positively and negatively fine-tuned HIF-1 in hypoxic hepatocarcinoma cells.

No MeSH data available.


Related in: MedlinePlus

NF-κB dual-regulates HIF-1α under short-term versus prolonged hypoxia(A) Vista predictions of NF-κB binding sites on the promoter of HIF1A. (B) ChIP analysis of the HIF1A promoter with NF-κB subunit antibodies and control IgG. (C,D) After HepG2 cells were transfected with shNC, shp65 or shcRel, and exposed to hypoxia for 4 h (H4) or 24 h (H24), the mRNA (C) and protein (D) levels of HIF-1α were assessed. The results were expressed as the mean±SEM of four independent experiments. *P < 0.05 and **P < 0.01 compared with shNC plus H4; #P < 0.05 compared with shNC plus H24. (E,F) After HepG2 cells were transfected with mock, p65 or cRel, and exposed to hypoxia for 4 h (H4) or 24 h (H24), the mRNA (C) and protein (D) levels of HIF-1α were assessed. The results were expressed as the mean±SEM of four independent experiments. *P < 0.05 and **P < 0.01 compared with mock plus H4; #P < 0.05 compared with mock plus H24.
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Figure 2: NF-κB dual-regulates HIF-1α under short-term versus prolonged hypoxia(A) Vista predictions of NF-κB binding sites on the promoter of HIF1A. (B) ChIP analysis of the HIF1A promoter with NF-κB subunit antibodies and control IgG. (C,D) After HepG2 cells were transfected with shNC, shp65 or shcRel, and exposed to hypoxia for 4 h (H4) or 24 h (H24), the mRNA (C) and protein (D) levels of HIF-1α were assessed. The results were expressed as the mean±SEM of four independent experiments. *P < 0.05 and **P < 0.01 compared with shNC plus H4; #P < 0.05 compared with shNC plus H24. (E,F) After HepG2 cells were transfected with mock, p65 or cRel, and exposed to hypoxia for 4 h (H4) or 24 h (H24), the mRNA (C) and protein (D) levels of HIF-1α were assessed. The results were expressed as the mean±SEM of four independent experiments. *P < 0.05 and **P < 0.01 compared with mock plus H4; #P < 0.05 compared with mock plus H24.

Mentions: Previous reports indicated that NF-κB enhanced HIF-1 expression and activation [15, 16]. Employing Vista Software [17], we found that both p50 and p65, but not c-Rel, were predicted to directly bind the promoter of HIF1A (Figure 2A). Chromatin immunoprecipitation (ChIP) assay also identified these NF-κB binding sites at 62162387-62162397 (for p50 and p65) and 62162718-62162728 (for p65) on the promoter of HIF-1A, respectively (Figure 2B).


Temporal regulation of HIF-1 and NF-κB in hypoxic hepatocarcinoma cells.

Jiang Y, Zhu Y, Wang X, Gong J, Hu C, Guo B, Zhu B, Li Y - Oncotarget (2015)

NF-κB dual-regulates HIF-1α under short-term versus prolonged hypoxia(A) Vista predictions of NF-κB binding sites on the promoter of HIF1A. (B) ChIP analysis of the HIF1A promoter with NF-κB subunit antibodies and control IgG. (C,D) After HepG2 cells were transfected with shNC, shp65 or shcRel, and exposed to hypoxia for 4 h (H4) or 24 h (H24), the mRNA (C) and protein (D) levels of HIF-1α were assessed. The results were expressed as the mean±SEM of four independent experiments. *P < 0.05 and **P < 0.01 compared with shNC plus H4; #P < 0.05 compared with shNC plus H24. (E,F) After HepG2 cells were transfected with mock, p65 or cRel, and exposed to hypoxia for 4 h (H4) or 24 h (H24), the mRNA (C) and protein (D) levels of HIF-1α were assessed. The results were expressed as the mean±SEM of four independent experiments. *P < 0.05 and **P < 0.01 compared with mock plus H4; #P < 0.05 compared with mock plus H24.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496226&req=5

Figure 2: NF-κB dual-regulates HIF-1α under short-term versus prolonged hypoxia(A) Vista predictions of NF-κB binding sites on the promoter of HIF1A. (B) ChIP analysis of the HIF1A promoter with NF-κB subunit antibodies and control IgG. (C,D) After HepG2 cells were transfected with shNC, shp65 or shcRel, and exposed to hypoxia for 4 h (H4) or 24 h (H24), the mRNA (C) and protein (D) levels of HIF-1α were assessed. The results were expressed as the mean±SEM of four independent experiments. *P < 0.05 and **P < 0.01 compared with shNC plus H4; #P < 0.05 compared with shNC plus H24. (E,F) After HepG2 cells were transfected with mock, p65 or cRel, and exposed to hypoxia for 4 h (H4) or 24 h (H24), the mRNA (C) and protein (D) levels of HIF-1α were assessed. The results were expressed as the mean±SEM of four independent experiments. *P < 0.05 and **P < 0.01 compared with mock plus H4; #P < 0.05 compared with mock plus H24.
Mentions: Previous reports indicated that NF-κB enhanced HIF-1 expression and activation [15, 16]. Employing Vista Software [17], we found that both p50 and p65, but not c-Rel, were predicted to directly bind the promoter of HIF1A (Figure 2A). Chromatin immunoprecipitation (ChIP) assay also identified these NF-κB binding sites at 62162387-62162397 (for p50 and p65) and 62162718-62162728 (for p65) on the promoter of HIF-1A, respectively (Figure 2B).

Bottom Line: Regulations between NF-κB and HIF-1 have not been adequately addressed in previous research.We also found that NF-κB p50 and p65 (RelA), but not c-Rel, bound the HIF-1a promoter, thus increasing its transcription.Dicer1, a key enzyme in miRNA biogenesis, was decreased by acute hypoxia but was later increased by HIF-1, rather than by the above-mentioned NF-κB subunits.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cancer, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China.

ABSTRACT
Regulations between NF-κB and HIF-1 have not been adequately addressed in previous research. Here, we report that hypoxia increased NF-κB in hepatocellular carcinoma cells. The HIF-1 protein level was rapidly induced by protein stabilization (by 2 hours) and then moderately decreased, whereas mRNA levels were reciprocally increased. We also found that NF-κB p50 and p65 (RelA), but not c-Rel, bound the HIF-1a promoter, thus increasing its transcription. In contrast, miR-199a-5p and miR-93, c-Rel downstream targets, decreased HIF-1α at both the mRNA and protein levels. Dicer1, a key enzyme in miRNA biogenesis, was decreased by acute hypoxia but was later increased by HIF-1, rather than by the above-mentioned NF-κB subunits. Thus, NF-κB both positively and negatively fine-tuned HIF-1 in hypoxic hepatocarcinoma cells.

No MeSH data available.


Related in: MedlinePlus