Use Of High-Throughput RNA Sequencing To Assess The Mechanism Of Functional Alteration In Human Trophoblast Cells In Response To Low-Dose Cadmium Exposure

Cadmium(Cd)is one of the heavy metal pollutants,which can accumulate in the human body and produce toxic effects on multiple tissues.Maternal Cd exposure may inhibit the development of the placenta and the fetus.However,the underlying molecular mechanism remains largely unknown.In this study,human placental trophoblast cell line HTR-8/SVneo was used as the experimental model,and the effects of different concentrations of Cd exposure on the proliferation and apoptosis of HTR-8/SVneo cells were detected by MTT,Brd U and TUNEL methods.Using RNA-sequencing(RNA-seq)technology,the differential expression genes(DEGs)in HTR-8/SVneo cells treated with2.5 μM Cd and 7.5 μM Cd were analyzed.These DEGs were functionally annotated by gene ontology(GO)database,combined with relevant literature reports,and were further confirmed by quantitative real-time PCR(q RT-PCR),and the functional roles of these genes in cell proliferation and apoptosis of the Cd-exposed cell were verified by RNA interference technology and cellular experiments.Cell functional experiment results showed that,compared with the control group,2.5μM-10μM Cd inhibited the proliferation of HTR-8/SVneo cells,and 7.5-10 μM Cd could induce significant apoptosis of HTR-8/SVneo cells.RNA-seq results showed that,79 and 526 genes were differentially expressed in 2.5 μM and 7.5 μM Cd-exposed groups when compared with the control group;while 378 genes were differentially expressed in7.5 μM Cd-exposed group when compared with 2.5 μM Cd-exposed group.Using GO annotation,literature mining and q RT-PCR confirmation,GSDMA was identified as one of the candidate genes inhibiting trophoblast cell proliferation when exposed to 2.5 μM Cd,and IGFBP-1 was identified as one of the candidate genes inducing trophoblast cell apoptosis when exposed to 7.5 μM Cd.The transfection of GSDMA si RNA could completely block the proliferation-inhibiting effect induced by Cd exposure;while the transfection of IGFBP-1 si RNA could completely block the apoptosis-promoting effect induced by Cd exposure.Based on these results,the following conclusions are drawn:(1)2.5 μM Cd exposure inhibited the proliferation of HTR-8/SVneo cells,while 7.5μM Cd exposure induced apoptosis in HTR-8/SVneo cells.(2)In the HTR-8/SVneo cells,the proliferation-inhibiting effects of 2.5 μM Cd exposure may be caused by the up-regulation of GSDMA expression.The apoptosis-promoting effects of 7.5 μM Cd exposure may be caused by the up-regulation of IGFBP-1.