Under hypoxic microenvironment, HIF-1 is known to play an important role in cellular adaptive response to hypoxia, and up-regulates various gene expressions related to angiogenesis, glycolysis, invasion and metastasis of cancer cells. Moreover, HIF-1 has been strongly associated with tumor radioresistance. So HIF-1 is an effective target for anticancer therapy. Until now, there are many kinds of HIF-1 inhibitors, but unfortunately there is no one which could inhibit HIF-1 activity with high efficiency.
To overcome the problem caused by HIF-1, it is critical to elucidate molecular mechanism behind the regulation of HIF-1 activity. Especially, finding novel genes responsible for the activation of HIF-1 is important, because we can exploit it as a therapeutic target to inhibit HIF-1 activity.
First we established a screening system to select novel genes which could up-regulate HIF-1 activity. We have already gotten several novel genes up-regulating HIF-1 activity. Gene X is one of them. How about the function of Gene X and the mechanism of up- HIF-1 activity?
We constructed an expression vector for Gene X, and performed a reporter gene assay to confirm that Gene X induces HIF-1 activity using our HIF- 1-dependentreporter gene 5HRE-Luc reporter gene, and also knockdown of Gene X resulted in the suppression of HIF-1 activity.
Using a different kind of reporter gene composed of oxygen-dependent degradation domain of HIF-1alpha and luciferase, we can monitor the stability of HIF-1alpha protein as luciferase bioluminescence. Overexpression of Gene X induced luciferase activity from the ODD-Luc fusion protein, and knockdown of Gene X significantly suppressed the luciferase activity. These results indicate that Gene X stabilizes HIF-1a protein.
Using western blotting and Luciferase activity assay to check HIF-1 protein level and luciferase activity , we found Gene X prolongs the half life of HIF-1.We confirmed that overexpression of Gene X increased HIF-1a expression level and knockdown of Gene X suppressed HIF-1a expression level.
Using real-time PCR, we found Gene X overexpression results in HIF-1a’s downstream genes such as VEGF,PDGF and bFGF2 mRNA expression increasing. Also we found Gene X knock down decreased HIF-1a’s downstream genes such as VEGF, PDGF and bFGF2 mRNA expression.
We established stable cancer cell lines constitutively expressing Gene X and knocked down Gene X to examined whether Gene X influences tumor growth or not. The results showed that overexpression of Gene X accelerated tumor growth after the transplantation, and tumor growth of Gene X knockdown cells was significantly delayed compared to control.
So far, we thought: Gene X induces HIF-1 activity, and stabilizes and prolongs half life of HIF-1a to accelerate the tumor growth; Knockdown of Gene X decrease HIF-1 activity and, decreases MVD and results in the tumor growth delay; Gene X might be a potential target for cancer therapy.
This project is supported by 973 key project (No:2011CB503704) and International Science & Technology Cooperation Project (No: 2010DFA31900) and National Natural Science Foundation of China (No:81272490, 60971055) .