| Objective:To investigate the effect of hypoxia inducible factor on neural tube defects and its possible mechanism.First, we established animal model of neural tube defects(NTDs)byretinoicacid. Embryonic brain from NTDs underwent a RNAsequencing(RNA-seq),bioinformatics analysis,found changes in the expression ofHIF3 a and BNIP3 in N TDs compared with the normal group. So HIF3 a and BNIP3 were usedas the main target genes of this study.Next, retinoic acid(RA) was added to cells cultured in vitro and the siRN A plasmid was transfected into the cells to interrupt the expression of HIF3 a,the mutual relations between the target genes HIF3 a,BNIP3 and cell apoptosis and the potential mechaniems in the development of NTDs were studied. Methods:1.When the mice were pregnant for 7 days(E7.5), a certain amount of retinoic acid(RA) was injected into the mice stomach, to prepare the NTDs model,then the embryonic brain from N TDs model mice at E10.5 was taken out to have a RNA sequencing and then the consequences were analyzed, it was found that the expressions of hypoxia inducible factor HIF1 a,HIF3a and the related downstream gene BNIP3 changed during the formation of NTDs.2.Detect the expression of HIF1 a,HIF3a,BNIP3 mRNA by Real-time PC R in embryonic brain from N TDs mice at E10.5.3.Adding 0 μM, 20μM, 40μM concentrations of retionic acid to PC12 cells,Real-time PCR and Western blotting were used to detect expressions of target genes in cells.4.After adding different concentrationsof retionic acid to cells,flow cytometry was used to detect cells apoptosis,the changes of reactive oxygen species(ROS) and mitochondrial membrane potential.5.The siRNA plasmid was transfected by liposome into the PC12 cells to interrupt the expression of HIF3 a,cells were devided into four groups: control group,retinoic acid intervention group,HIF3α siRNA group and retinoic acid plus HIF3α siRNA group,Real-time PCR and Western blotting were used to detect the expression of BNIP3.6.Cells apoptosis of four groups were detected by flow cytometry.7.Caspase3 staining was performed on four groups,caspase3 activation was observed under fluorescence microscope. Results:1.Animal NTDs model was successfully induced by using RA of 21 mg/kg,the outline and segment of embryos from control group are clear,the brain is plump and the volume is big. By contrast,the embryo of the abnormal group is smaller,segment is not clear as control group and even encephalocele,the result of sequencing analysis showed that the expression of HIF1 a is decreased but HIF3 a and BNIP3 are increased.2.Real-time PCR results was consistent with the results of RNA-seq, the expression level of HIF1 a was decreased and HIF3 a and BNIP3 were increased,which indicated HIF3 a and BNIP3 may be associated with NTDs.3.Real-time PCR and Western blotting results indicated that with the retinoic acid dosage increased,the expression levels of HIF3α and BNIP3 increased too.4.Flow cytometry results indicated that with the retinoic acid dosage increased,the apoptosis cells increased,more reactive oxygen species were produced,the cell mitochondrial membrane potential decreased.5.The transfection efficiency of the siRN A is about 70%. Interrupting the expression of HIF3 a,hindered the expression of BNIP3 and the rise of the Bnip3 expression inducted by retinoic acid.6.Flow cytometry results indicated that the interference of HIF3α expression inhibited apoptosis induced by retinoic acid.7. Retinoic acid can acvitate caspase3,HIF3 a interference can inhibit caspase3 activation. Conclusion:Retinoic acid induces HIF3 aexpression increasing and acvitates caspase3, HIF3 a regulates the expression of BNIP3 and affects cell apoptosis. |
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