Effects Of Cadmium On Toxicity,Migration And Invasion Of The Human Trophoblast Cells HTR-8/SVneo And Its Mechanisms

Cadmium is the most common heavy metal pollutant in the environment.It is a non-essential element of the human body and can cause damage to multiple systems and multiple organs.It affects seriously human health.Cadmium exposure during pregnancy can lead to adverse pregnancy outcomes,such as abortion,intrauterine growth retardation and pre-eclampsia.Therefore,in order to investigate the mechanisms of cadmium-induced adverse pregnancy outcomes at the cellular level.In this study,the human first-trimester trophoblast cells HTR-8/SVneo were selected as object of study.After treatment with Cd Cl₂,MTT assay,cell morphology observation,flow cytometry,transwell assay,gelatin zymography and RNA-seq were used to study the effects of cadmium on toxicity,migration and invasion of HTR-8/SVneo cells.The toxicity mechanism of cadmium was also explored.The results were as follows:（1）Cadmium has a significant toxicity on HTR-8/SVneo cells.MTT assay showed that the cells viability decreased significantly with increasing concentrations of Cd Cl₂and treatment time（P<0.01）.It exhibited a dose-time-effect relationship.Phase contrast microscopy showed that the cells adhesion decreased gradually with increasing concentrations of Cd Cl₂.Transmission electron microscopy showed that the cells had nucleus pyknosis,and vacuoles,mitochondrial swelling and mitochondrial ridge disappeared in the cytoplasm.RNA-seq showed that there were 7270 significant differential genes.4112 genes were significantly up-regulated.3158 genes were significantly down-regulated.10 KEGG pathways and 377 GO functions were significantly enriched.The number of high-impact mutation site in the gene sequence was significantly increased.（2）Cadmium can induce oxidative stress in HTR-8/SVneo cells and causes oxidative damage in cells.After HTR-8/SVneo cells were treated with Cd Cl₂,the results of oxidative stress indicators were as follows:The ROS level increased gradually with increasing concentrations of Cd Cl₂.It reached a very significant level at high concentrations（P<0.01）.The MDA content increased gradually with increasing concentrations of Cd Cl₂.It reached a very significant level at high concentrations（P<0.01）.The GSH content increased first and then decreased gradually with increasing concentrations of Cd Cl₂.It was the highest when Cd Cl₂ concentration was 25μmol/L（P<0.01）.It was the lowest when Cd Cl₂ concentration was 200μmol/L（P<0.05）.The GSH-Px activity and the SOD activity increased gradually with increasing concentrations of Cd Cl_2.It reached a very significant level at high concentrations（P<0.01）.The CAT activity increased first and then decreased slowly with increasing concentrations of Cd Cl₂.It was the highest when Cd Cl₂ concentration was 50μmol/L（P<0.01）.It increased significantly when the concentration of Cd Cl₂ was 100 and 200 umol/L（P<0.01）.（3）Cadmium can induce apoptosis of HTR-8/SVneo cells.The results of flow cytometry showed that the apoptosis rate of HTR-8/SVneo cells increased gradually with increasing concentrations of Cd Cl₂（P<0.01）.It exhibited a dose-effect relationship.The results of apoptosis pathway were as follows:The mitochondrial membrane potential of HTR-8/SVneo cells decreased gradually with increasing concentrations of Cd Cl₂（P<0.01）.The Caspase-3 activity decreased first and then increased with increasing concentrations of Cd Cl_2.It decreased when the concentration of Cd Cl₂ was 25 and 50μmol/L（P<0.05 at 50μmol/L）.It increased significantly at 100 and 200μmol/L（P<0.05）.The Caspase-9 activity increased gradually first and then decreased with increasing concentrations of Cd Cl₂.It was the highest when the concentration of Cd Cl₂ was 100μmol/L（P<0.01 at 50 and 100μmol/L）.The intracellular Ca²⁺concentration increased with increasing concentrations of Cd Cl₂.It increased slightly when the concentration of Cd Cl₂ was 25 and 50μmol/L.It increased significantly when the concentration of Cd Cl₂was 100 and 200μmol/L（P<0.01）.The Fas/Fas L genes were significantly up-regulated after HTR-8/SVneo cells treated with Cd Cl₂.（4）Cadmium can decrease the migration and invasion of HTR-8/SVneo cells.Transwell assay showed that the migration and invasion of HTR-8/SVneo cells decreased with increasing concentrations of Cd Cl₂（P<0.01）.It exhibited a dose-effect relationship.The results of the invasion mechanism were as follows:Gelatin zymography showed that Cd Cl₂ treatment had no significant effect on MMP-2 activity,but inhibited MMP-9activity.The balance of MMPs/TIMPs genes expression was broken after HTR-8/SVneo cells treated with Cd Cl_2.In conclusion,cadmium has significant toxicity on HTR-8/SVneo cells.It can reduce cell viability and cell adhesion.It damages cell nuclei and organelles.It affects the transcriptome,resulting in a large number of differential genes.Cadmium induces oxidative stress in HTR-8/SVneo cells,causing oxidative damage to cells.It may induce apoptosis through mitochondrial pathway,endoplasmic reticulum stress pathway and death receptor pathway.At the same time,cadmium can also reduce the migration and invasion of HTR-8/SVneo cells.The inhibition of cadmium on invasion is related to cadmium inhibiting MMP-9 activity and cadmium breaking MMPs/TIMPs gene expression balance.