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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

HIF-1 upregulates Dicer1 and miR-93/199a-5p under hypoxia(A) Vista prediction of HIF-1 binding sites on the promoter of Dicer1. The pink arrow shows the HRE sequence. (B) ChIP analysis of the Dicer1 promoter with NF-κB subunit antibodies and control IgG. (C,D) After HepG2 cells were transfected with shNC or shHIF1A, and exposed to hypoxia for 0 h (N), 4 h (H4) or 24 h (H24), the mRNA (C) and protein (D) levels of HIF-1α, p50, p65, cRel, and Dicer1, as well as the expressions of miR-93 and miR-199a-5p (C) were assessed. The results of (C) were expressed as the means of four independent experiments and normalized against shNC plus N. (E) The regulatory network involving HIF-1, NF-κB, Dicer1 and miRNAs under hypoxia.
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Figure 6: HIF-1 upregulates Dicer1 and miR-93/199a-5p under hypoxia(A) Vista prediction of HIF-1 binding sites on the promoter of Dicer1. The pink arrow shows the HRE sequence. (B) ChIP analysis of the Dicer1 promoter with NF-κB subunit antibodies and control IgG. (C,D) After HepG2 cells were transfected with shNC or shHIF1A, and exposed to hypoxia for 0 h (N), 4 h (H4) or 24 h (H24), the mRNA (C) and protein (D) levels of HIF-1α, p50, p65, cRel, and Dicer1, as well as the expressions of miR-93 and miR-199a-5p (C) were assessed. The results of (C) were expressed as the means of four independent experiments and normalized against shNC plus N. (E) The regulatory network involving HIF-1, NF-κB, Dicer1 and miRNAs under hypoxia.

Mentions: We next questioned whether the elevation of Dicer1 and candidate miRNAs occurs due to regulation by the transcriptional factors HIF-1 and NF-κB. Using Vista software, a binding site of HIF-1 including a hypoxia responsible element (HRE) encoding 5′-GCGTC-3′ was predicted in the promoter of Dicer1 (Figure 6A). No potential binding sites in the Dicer1 promoter were predicted for p50, p65 and c-Rel, which was also confirmed by the ChIP assay (Figure 6A and 6B). Moreover, the binding sites for HIF-1 were also predicted in the promoters of p50, p65 and c-Rel (Figure S5). Indeed, knockdown of HIF-1α significantly reduced the expressions of p50, p65, c-Rel, Dicer1, miR-93, and miR-199a-5p under hypoxia (Figure 6C and 6D). Together, these results suggested that NF-κB temporally modulates HIF-1 in the short-term versus prolonged hypoxia (Figure 6E).


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)

HIF-1 upregulates Dicer1 and miR-93/199a-5p under hypoxia(A) Vista prediction of HIF-1 binding sites on the promoter of Dicer1. The pink arrow shows the HRE sequence. (B) ChIP analysis of the Dicer1 promoter with NF-κB subunit antibodies and control IgG. (C,D) After HepG2 cells were transfected with shNC or shHIF1A, and exposed to hypoxia for 0 h (N), 4 h (H4) or 24 h (H24), the mRNA (C) and protein (D) levels of HIF-1α, p50, p65, cRel, and Dicer1, as well as the expressions of miR-93 and miR-199a-5p (C) were assessed. The results of (C) were expressed as the means of four independent experiments and normalized against shNC plus N. (E) The regulatory network involving HIF-1, NF-κB, Dicer1 and miRNAs under hypoxia.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: HIF-1 upregulates Dicer1 and miR-93/199a-5p under hypoxia(A) Vista prediction of HIF-1 binding sites on the promoter of Dicer1. The pink arrow shows the HRE sequence. (B) ChIP analysis of the Dicer1 promoter with NF-κB subunit antibodies and control IgG. (C,D) After HepG2 cells were transfected with shNC or shHIF1A, and exposed to hypoxia for 0 h (N), 4 h (H4) or 24 h (H24), the mRNA (C) and protein (D) levels of HIF-1α, p50, p65, cRel, and Dicer1, as well as the expressions of miR-93 and miR-199a-5p (C) were assessed. The results of (C) were expressed as the means of four independent experiments and normalized against shNC plus N. (E) The regulatory network involving HIF-1, NF-κB, Dicer1 and miRNAs under hypoxia.
Mentions: We next questioned whether the elevation of Dicer1 and candidate miRNAs occurs due to regulation by the transcriptional factors HIF-1 and NF-κB. Using Vista software, a binding site of HIF-1 including a hypoxia responsible element (HRE) encoding 5′-GCGTC-3′ was predicted in the promoter of Dicer1 (Figure 6A). No potential binding sites in the Dicer1 promoter were predicted for p50, p65 and c-Rel, which was also confirmed by the ChIP assay (Figure 6A and 6B). Moreover, the binding sites for HIF-1 were also predicted in the promoters of p50, p65 and c-Rel (Figure S5). Indeed, knockdown of HIF-1α significantly reduced the expressions of p50, p65, c-Rel, Dicer1, miR-93, and miR-199a-5p under hypoxia (Figure 6C and 6D). Together, these results suggested that NF-κB temporally modulates HIF-1 in the short-term versus prolonged hypoxia (Figure 6E).

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