PAX3 regulates ferroptosis in trophoblast cells under hypoxia
Objective To investigate the mechanism of paired box gene(PAX)3 regulating ferroptosis in trophoblast cells under hypoxia.Methods The experiment was conducted from January to December 2023 at the Institute of Eugenics Medicine,the Affiliated Women's Hospital of Jiangnan University.Human chorionic trophoblast cells(HTR-8/SVneo)were cultured in vitro under normoxia of 20％oxygen and hypoxia of 2％oxygen,respectively,HTR-8a/Svneo cells were divided into 7 groups according to different treatments:normoxia group,hypoxia group,hypoxia+NC plasmid group,hypoxia+PAX3(OE)group,hypoxia+SLC40A1(OE)group,hypoxia+VDAC3(OE)group,hypoxia+PAX3(OE)+VDAC3(KD)group.Plasmid transfection technology was used to knockdown VDAC3 and over-express PAX3,SLC40A1,and VDAC3 to verify the regulation of PAX3 on SLC40A1 and VDAC3 expressions in HTR-8/SVneo cells.The mitochondria in HTR-8/SVneo cells under normoxia,normoxia+Erastin,and hypoxia conditions were observed by electron microscopy.Cell death rate was detected with live/dead cell double staining kit.Immunofluorescence cytochemical staining was used to detect the localization and expressions of PAX3,SLC40A1,and VDAC3 in trophoblast cells of each group.The mRNA and protein levels of PAX3,SLC40A1,and VDAC3 in each group were detected by real-time quantitative PCR and Western blot.Independent sample t test was used.Results(1)The mitochondrial morphology of cells exposed to hypoxia resembled that observed under normoxia+Erastin treatment.Notably,there was a significant reduction in mitochondrial shrinkage and volume,an increase in membrane electron density,as well as a decrease and blurring of the inner ridge.(2)Under hypoxia,the live/dead double staining kit showed that the cell death rate was increased.Immunofluorescence results showed that PAX3 was mainly expressed in the nucleus,SLC40A1 was mainly expressed in the cytoplasm,and VDAC3 was mainly expressed in the mitochondria.(3)Compared with the normoxia group,the cell death in the hypoxia group increased,and the mRNA and protein expressions of PAX3 and SLC40A1 decreased in the HTR-8a/Svneo hypoxia group,but the mRNA and protein expressions of VDAC3 increased in the HTR-8a/Svneo hypoxia group.After knocking down PAX3,the expression of SLC40A1 was decreased,and the cells died extensively.After over-expressing PAX3,the expression of SLC40A1 was increased,and the cell survival rate increased.Conclusions Hypoxia induces ferroptosis in trophoblast cells.PAX3 affects SLC40A1 expression by regulating ferroptosis in trophoblast cells,but the knockdown or over-expression of PAX3 has no effect on the expression of VDAC3.
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维普
