Construction Of Recombinant Adenoviral Expressed Vector DMT1 And Its Functional Study

Divalent metal transporter 1 (DMT1), previously known as natural resistance associated macrophage protein 2 (Nramp2) , is a mammalian transmembrane iron transporter which transfers iron across the apical surface of intestinal cells and out of transferrin cycle endosomes. The mRNA of DMT1 has two forms, DMT1+IRE and DMT1-IRE form. The DMT1+IRE form transcript contains a canonical iron-responsive element (IRE) in the 3' untranslated region, whereas that of DMT1-IRE form lacks the IRE motif. DMT1 is made of twelve domaims and domain four is most important for its function. Researchers have proved that DMT1 is widely expressed in the brain. Its abnormal expression in some areas of the brain may change the iron metabolism of the brain and is related to the iron accumulation of some neurodegenerative diseases. In order to study the iron uptaking effect of DMT1 domain four, we constructed recombinant adenoviral expressed vector carrying DMT1 domain four and then infected glioma C6 cells. Glioma C6 cells were infected with recombinant adenoviruses vAd-DMT1+IRE and vAd-DMT1-IRE. The expression of target gene was tested by RT-PCR and Western Blotting. After the infected glioma C6 cells were treated with FeSO₄, the intracellular iron contents were detected by inductively coupled plasma-mass spectrometry (ICP-MS). The effect on cell viability and cell apoptosis of glioma C6 cell was analyzed by MTT method and detection of DNA fragmentation. The intracellular SOD,GSH-px,OH·and MDA were measured. The ultrastructure was observed under electron microscope. The results were as following:1.Total RNA was isolated by using Trizol Reagent from frozen tissues of human duodenum according to the manufacturer's instructions. The human DMT1 domain four gene was amplified by RT-PCR. The product of PCR is 380bp, which is confirmed by sequencing identification.2. The human DMT1 domain four gene was clonded to adenovial shuttle plasmid pAdTrack-CMV. Then the resultant pAdTrack-CMV-DMT1-4 was cotransfected into BJ5183 bacteria with the plasmid pAdeasy-1. The adenoviral plasmid carrying DMT1-4 was generated with homologous recombinant in bacteria3. The adenoviruses were transfected in 293 cells. The recombinant adenovirus vAd-DMT1-4, which has stable infectivity, was packed in 293 cells.4. Glioma C6 cells were infected with recombinant adenoviruses vAd-DMT1-4, the increase of GFP expression was time dependent and GFP expression reached good infection efficiency at 36h. The expression of target gene was tested by RT-PCR, and the result showed that the target gene expression significantly increased (P＜0.05) in infected vAd- DMT1-4 glioma C6 cell group compared with normal cells group.5. Infected glioma C6 cells with recombinant adenoviruses vAd-DMT1-4 were treated with 1mM FeSO4(containing Vc) for 30min and 60min respectively. The intracellular iron contents were detected by inductively coupled plasma-mass spectrometry (ICP-MS). The results showed that There was no significant difference in iron contents in infected vAd- DMT1-4 group when compared with either control group or GFP(vector control) group.6. Glioma C6 cells were infected with recombinant adenoviruses vAd-DMT1+IRE and vAd-DMT1-IRE. GFP expression increased time dependently. The expression of target gene was tested by RT-PCR, and the data showed that the expression of target gene increased significantly in infected vAd-DMTI+IRE group and vAd-DMT1-IRE group compared with normal cells group and GFP group (P＜0.05).7. The expression of target gene was tested by Western Blotting, data showed that it increased significantly in infected vAd-DMT1+IRE group and vAd-DMT1-IRE group compared with normal cells group and GFP group (P＜0.05).8. Infected glioma C6 cells were treated with 1mM FeSO₄(containing Vc) for 30min and 60min respectively. The intracellular iron contents were detected by ICP-MS. The results showed that DMT1+IRE and DMT1-IRE transported iron, the iron contents increased significantly compared with that of control group and GFP group (P＜0.01).9. The viability of glioma C6 cells was inhibited in infected vAd-DMT1+IRE and vAd-DMT1-IRE compared with control group (P＜0.05), while there was no significant difference between the normal cells group and GFP group.10. Analysis of DNA extracted from vAd-DMT1+IRE and vAd-DMT1-IRE infected cells showed the typical "ladder pattern", indicating the formation of mono- and oligonucleosomes.11. The amounts of hydroxyl free radicals (·OH) and MDA were both significantly increased in vAd-DMT1+IRE and vAd-DMT1-IRE infected cells compared with that of GFP group (P＜0.01) .12. The activities of SOD and GSH-px were both significantly decreased in vAd-DMT1+IRE and vAd-DMT1-IRE infected cells compared with that of GFP group (P＜0.05). 13. Mitochondria swelling, vacuolization, no mitochondrial cristae, heterochromatin increase and apoptosis were observed under electron microscope in vAd-DMT1 +IRE and vAd-DMT1-IRE infected cells, while the cells in GFP group were not damaged.The results suggest that recombinant DMT1+IRE and DMT1-IRE could transport ferrous iron, while DMT1-4 couldn't. Increased DMT1 expression induced iron accumulation in glioma C6 cells. Iron accumulation inhibited cell growth and induced cell apoptosis by generating high level OH·and decreasing the activities of SOD and GSH-px inducing oxidative stress. Our study may provide experimental evidence for research on iron transportation and the neuron toxicity of high iron concentration in the brain.