C20orf20 Is Involved In The Pathogenesis Of Preeclampsia By Regulating The Proliferation And Migration Of Trophoblast Cells

Research Background and Objective:Preeclampsia is a pregnancy-specific disorder characterized clinically by elevated blood pressure and albuminuria that is associated with maternal mortality and adverse neonatal/fetal outcomes,including perinatal maternal death,preterm birth and intrauterine growth restriction.The etiology and pathogenesis of the disease have not been fully elucidated.At present,most experts and scholars believe that the occurrence of preeclampsia may be closely related to the biological dysfunction of placental trophoblast cells.During early gestation,trophoblast cells invade to reshape the uterine spiral artery into a low-resistance vascular channel with maximum dilation,thus ensuring high blood flow to the uterine placenta.Impaired biological capacity of the placental trophoblast can lead to stenosis of the spiral arteries of the uterus and reactive blood vessels and consequent placental dysfunction,leading to the progression of preeclampsia.With the continuous development of scientific research technology and molecular medicine,the related expression and regulatory mechanism of Chromosome 20open reading frame 20（C20orf20）in various cancers were constantly clarified.It has been gradually confirmed that C20orf20 gene plays an important role in regulating metabolic and immune functions of pathological changes in the body.However,the relationship between C20orf20 gene and the proliferation,migration and invasion of placental trophoblast cells and the occurrence and development of preeclampsia has not been reported.A previous proteomics study on preeclampsia conducted by my research team found that C20orf20protein was significantly down-regulated in the placental tissue of preeclampsia pregnant women.It was speculated that down-regulated C20orf20 protein could inhibit the proliferation and invasion of placental trophoblast cells.Resulting in placental trophoblast cells to endometrial and vascular infiltration of insufficient,preeclampsia.Therefore,this study will clarify the role of C20orf20 gene in the biological dysfunction of trophoblastic cells,help to improve the pathogenesis theory of preeclampsia,provide potential therapeutic targets for preeclampsia and improve its adverse pregnancy outcomes,and provide relevant experimental evidence.Materials and Methods:1.Human chorionic trophoblast cells HTR-8/Svneo required for the experiment were donated by Professor Wang Haibin of Xiamen University and kept in our laboratory.2.Lentivirus transfection method was used to construct human chorionic trophoblast cell（HTR-8/Svneo）stable cell lines with C20orf20 gene overexpression and knockdown expression.3.The trophoblast cells were cultured using RPMI-1640 medium containing 10%fetal bovine serum and 100U/m L penicillin/streptomycin double antibody,and a cell incubator at37℃and 5%CO₂.4.C20orf20 gene overexpression and knockdown trophoblast trophoblast cell lines and their respective control plasmid trophoblast trophoblast cell lines were inoculated into 96-well plates,respectively,and cell proliferation was detected by CCK8 cell proliferation detection kit 24 h after culture.5.C20orf20 gene overexpression and knockdown trophoblast trophoblast cell lines and their respective control plasmid trophoblast trophoblast cell lines were inoculated into 6-well plates,respectively,and apoptosis was detected by flow cytometry using Annexin V-APC/7-AAD double staining after 24 culture.The cell cycle was detected by flow cytometry using PI staining 24 h after culture.6.C20orf20 gene overexpression and knockdown trophoblast trophoblast cell lines and their respective control plasmid trophoblast trophoblast cell lines were inoculated into 24 well plates,respectively,and Transwell experiment was conducted to verify the invasion ability of trophoblast cells in each group after stabilization.Results:1.HTR-8/Svneo cells transfected with C20orf20 gene overexpression,knockout and stable transmutation strains in the control group were cultured.2.The results of cell proliferation experiment showed that although overexpression of C20orf20 gene stabilized HTR-8/Svneo cell line,the cell proliferation ability of HTR-8/Svneo cell line was not significantly increased,but C20orf20 gene knockout group significantly reduced the cell proliferation ability of HTR-8/SVneo cell line.3.Flow cytometry analysis of cell cycle showed that compared with the control HTR-8/Svneo cell line group,the proportion of cells in S phase was significantly higher in C20orf20 gene knock-down stable transfer HTR-8/Svneo cell line group,while the proportion of cells in G2/M phase was decreased.The results indicated that C20orf20 knockdown inhibited HTR-8/Svneo cells from entering G2/M phase from S phase.4.The results of apoptosis experiment showed that compared with the control HTR-8/Svneo cell lines,the number of apoptosis and death cells in HTR-8/Svneo cell lines overexpressing C20orf20 was reduced.The number of apoptosis and death cells in the C20orf20 knockdown stable HTR-8/Svneo strain increased significantly.5.The results of cell invasion experiment showed that the invasion ability of C20orf20knockdown stable HTR-8/Svneo cell line was decreased.Conclusion:C20orf20 gene knockdown significantly reduced the proliferation and invasion ability of trophoblast cells.It affected the cell cycle of trophoblast cells and inhibited trophoblast cells from entering G2/M phase from S phase.The apoptosis and death of trophoblast cells were promoted.In conclusion,C20orf20 gene is likely to be involved in the pathogenesis of preeclampsia by influencing the cellular biological functions of placental trophoblast cells.