Effect Of Hypoxia On The Iron Regulatory Proteins 1 (irp1) Expression And The Mechanisms Involved In Hepg2 Cells

Objective The study was designed to research the effects of hypoxia on the transcription of iron regulatory proteins 1 (IRP1) in human hepatoma HepG2 cells and explore the regulatory mechanism.Methods The HepG2 cells were exposed to hypoxia (1% O₂) or 400αM CoCl2 for 6 h, the IRP1 mRNA transcription was analyzed by RT-PCR,and the total proteins were extracted to detect the expression of IRP1 and hypoxia inducible factor-1αααHIF-1ααby Western blotting. Simultaneously,The effect of different duration of hypoxia on IRP1 mRNA transcription was detected by QRT-PCR. Using the online data software MatInspector, the presence of putative hypoxia responsive element (HRE) of IRP1 were revealed. The binding of putative HRE of IRP1 with HIF-1 was analyzed by electrophoretic mobility shift assay (EMSA) and supershift assay. The putative HRE core sequence was subcloned into luciferase reporter vector and then transiently transfected into HepG2 cells. After hypoxia treatment, the luciferase activities of samples were measured to verify the existence of HRE in IRPs. The core sequence of HRE (RCGTG) was site-directed mutated to (RAATG) and constructed into luciferase reporter vectors. The luciferase activity was measured under normoxia or hypoxia after transfecting. Furthermore, HIF-1αexpressed plasmid was cotransfected into cells with the vectors containing natural or mutated HRE sequence. The cells were cultured in normoxia and the luciferase activity was quantitated to identify the relationship between HIF-1 and transcription of IRP1.Results The content of HIF-1αin HepG2 was visibly increased after hypoxia or CoCl2 treatment for 6 h, while the expression of IRP1 remarkably decresed. The transcriptional level of IRP1 mRNA was also down-regulated. The transcriptional level of IRP1 mRNA persistently decreased before 6 h time point for hypoxia, but increased in hypoxic condition for more than 6 h. Four putative HRE were found by MatInspector which respectively existed in the sequences of -21756^-21740,-21251^-21235,-20754^-20738 and -20753^-20737 of 5'-regulatory region of IRP1 gene(set the translation initiation site as +1. The -21756^-21740 sequence (HRE1)was confirmed to specific bind with HIF-1 through the analysis and exclusion by EMSA and supershift. The luciferase activites with the plasmids containing HRE1 sequence in HepG2 cells markedly decreased under hypoxia compared with that of normoxia. Furthermore, the luciferase activities of HepG2 cells with the constructs, which had no putative HRE1 or contained mutational HRE1 sequence, had not obvious change whether in hypoxia or normoxia. The data with cotransfected vectors showed that the luciferase activities of cells with HRE1 was markedly lower than that of cells with empty vector.Conclusions Hypoxia within 6 h could down-regulate the transcription of IRP1 through the binding of HIF-1 with the functional HRE in the 5'-regulatory region of IRP1, which may inhibite the mRNA and protein level of IRP1.