Research Paper Volume 13, Issue 1 pp 364—375

Enhancer-bound Nrf2 licenses HIF-1α transcription under hypoxia to promote cisplatin resistance in hepatocellular carcinoma cells

Nrf2 binds to a HIF-1A enhancer element in a hypoxia-insensitive manner. (A) HIF-1α mRNA levels were not changed by hypoxia in HepG2 cells. HepG2 cells were incubated under hypoxic condition (5% O2) for indicated time. mRNA was prepared and RT-qPCR was performed with HIF-1A specific primers. Student’s t-test was performed to evaluate the statistical significance: ns, not significant. (B) HIF-1α mRNA was up-regulated by hypoxia in HepG2 cells. Experiments were performed as in (A). Student’s t-test was performed to evaluate the statistical significance. *PC) Hypoxia-induced HIF-1α mRNA expression was Nrf2-dependent in HepG2/DDP cells. HepG2/DDP cells were transfected with Nrf2-specific siRNA then incubated under mild hypoxic condition (5% O2) for indicated time. RT-qPCR was performed as in (A). Student’s t-test: *PD) Nrf2 hyper-activation increased HIF-1α mRNA expression in HepG2 cells under hypoxia. HepG2 cells were transfected with KEAP1-specific siRNA then incubated with 5% O2 for indicated time points. RT-qPCR was performed as in (A). Student’s t-test: ***PE) Diagram showing the conserved Nrf2 binding site at the 5’-end of HIF-1α gene. This site has been shown to be bound by Nrf2 and regulate HIF-1α expression before. (F) Nrf2 binding to HIF-1α enhancer stronger in HepG2/DDP than HepG2 cells and was not sensitive to hypoxia. HepG2 and HepG2/DDP cells were chromatin-immuno-precipitated (ChIP) by using Nrf2-specific antibody. The binding of the conserved site shown in (E) was detected by RT-qPCR. Student’s t-test: ***PG) Confirming Nrf2 binding to HIF-1α enhancer by regular PCR. Indicated ChIP samples from (F) were amplified with specific primers to the conserved Nrf2 binding site by regular PCR then subject to DNA electrophoresis.

Figure 4. Nrf2 binds to a HIF-1A enhancer element in a hypoxia-insensitive manner. (A) HIF-1α mRNA levels were not changed by hypoxia in HepG2 cells. HepG2 cells were incubated under hypoxic condition (5% O2) for indicated time. mRNA was prepared and RT-qPCR was performed with HIF-1A specific primers. Student’s t-test was performed to evaluate the statistical significance: ns, not significant. (B) HIF-1α mRNA was up-regulated by hypoxia in HepG2 cells. Experiments were performed as in (A). Student’s t-test was performed to evaluate the statistical significance. *P<0.01, **P<0.001. (C) Hypoxia-induced HIF-1α mRNA expression was Nrf2-dependent in HepG2/DDP cells. HepG2/DDP cells were transfected with Nrf2-specific siRNA then incubated under mild hypoxic condition (5% O2) for indicated time. RT-qPCR was performed as in (A). Student’s t-test: *P<0.01, **P<0.001. (D) Nrf2 hyper-activation increased HIF-1α mRNA expression in HepG2 cells under hypoxia. HepG2 cells were transfected with KEAP1-specific siRNA then incubated with 5% O2 for indicated time points. RT-qPCR was performed as in (A). Student’s t-test: ***P<0.0001. (E) Diagram showing the conserved Nrf2 binding site at the 5’-end of HIF-1α gene. This site has been shown to be bound by Nrf2 and regulate HIF-1α expression before. (F) Nrf2 binding to HIF-1α enhancer stronger in HepG2/DDP than HepG2 cells and was not sensitive to hypoxia. HepG2 and HepG2/DDP cells were chromatin-immuno-precipitated (ChIP) by using Nrf2-specific antibody. The binding of the conserved site shown in (E) was detected by RT-qPCR. Student’s t-test: ***P<0.0001, ns, not significant. (G) Confirming Nrf2 binding to HIF-1α enhancer by regular PCR. Indicated ChIP samples from (F) were amplified with specific primers to the conserved Nrf2 binding site by regular PCR then subject to DNA electrophoresis.