PAX3在缺氧状态下调控滋养细胞铁死亡的机制研究

PAX3 regulates ferroptosis in trophoblast cells under hypoxia

目的探讨配对盒基因(PAX)3在缺氧状态下调控滋养细胞铁死亡的机制。方法该实验于2023年1月至12月在江南大学附属妇产医院优生优育医学研究所完成。分别在20%氧气的常氧状态下和2%氧气的缺氧状态下体外培养人绒毛膜滋养层细胞(HTR-8/SVneo),将HTR-8a/Svneo细胞按照不同的处理分为7组:常氧组、缺氧组、缺氧+NC质粒组、缺氧+PAX3(OE)组、缺氧+SLC40A1(OE)组、缺氧+VDAC3(OE)组、缺氧+PAX3(OE)+VDAC3(KD)组。运用质粒转染技术敲低电压依赖性阴离子通道亚型3(VDAC3)和过表达PAX3、SLC40A1、VDAC3来验证细胞中PAX3调控SLC40A1、VDAC3的表达。使用电子显微镜观察常氧状态、常氧+铁死亡诱导剂(Erastin)、缺氧状态下HTR-8/SVneo细胞线粒体情况。用活/死细胞双染色试剂盒检测细胞死亡率。采用免疫荧光细胞化学染色检测各组滋养细胞PAX3、SLC40A1、VDAC3的定位及表达。实时定量PCR法和Western blot法检测各组细胞PAX3、SLC40A1、VDAC3 mRNA和蛋白表达。使用独立样本t检验。结果(1)缺氧状态下的细胞线粒体结构与常氧状态+Erastin的线粒体结构相似,线粒体皱缩、体积减小,膜电子密度增高,内脊减少、模糊。(2)缺氧状态下,活/死细胞双染色试剂盒的结果显示细胞死亡率增加。免疫荧光结果显示细胞PAX3主要在胞核内表达,SLC40A1主要在胞浆内表达,VDAC3主要在线粒体中表达。(3)与常氧状态相比,缺氧处理组细胞死亡增多,HTR-8a/Svneo缺氧组中PAX3、SLC40A1 mRNA和蛋白表达量均降低;HTR-8a/Svneo缺氧组中VDAC3 mRNA和蛋白表达升高;敲低PAX3后,SLC40A1表达降低,细胞大量死亡;过表达PAX3后,SLC40A1表达增高,细胞存活率升高。结论缺氧会诱导滋养细胞铁死亡。PAX3通过调控滋养细胞铁死亡影响SLC40A1表达,但PAX3的敲除或过表达对VDAC3表达没有影响。

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.