title: Effects of shRNA Targeting Maspin on the Invasion of Extravillous Trophoblast Cell.
abstract: ObjectiveHypomethylation of the maspin gene results in increased expression of maspin in preeclamptic placentas. However, maspin gene function and the molecular aspects in placentation remain largely unclear. The study was designed to investigate the effects of maspin on the invasion of extravillous trophoblast cell line (TEV-1) and the molecular mechanism.Study DesignWe cloned short hairpin RNA (shRNA) targeting maspin gene into plasmid pGenesil-1.1 eukaryotic expression vector and then transfected it using adenovirus. The methylation rates in the maspin gene were detected by bisulfite sequencing polymerase chain reaction; the invasive ability of trophoblast cells was examined by Transwell chamber assay; the mRNA and protein expression of maspin and some invasive related gene was detected by reverse transcription-polymerase chain reaction and Western blot analysis.ResultsAfter the maspin expression was successfully knocked down, the methylation rates in the maspin gene were significantly increased, and the number of cells invading through Matrigel (Corning Life Sciences and BD Biosciences) was obviously increased. The mRNA levels of vascular endothelial growth factor-A (VEGF-A), vascular endothelial growth factor-C (VEGF-C), and matrix metalloproteinase-2 (MMP2) were increased significantly.ConclusionUsing shRNA technology, this study further verified that maspin gene methylation could decrease maspin expression and inhibit the invasion of TEV-1 cells through VEGF-A, VEGF-C, and MMP2.
authors.affiliation: Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.
authors.forename: Xinwei
authors.initials: X
authors.lastname: Shi
authors.affiliation: Department of Obstetrics, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Liu
authors.affiliation: Department of Urology, Wuhan Third Hospital, Guanggu on Campus, Wuhan, Hubei, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Liu
authors.affiliation: Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.
authors.forename: Dongrui
authors.initials: D
authors.lastname: Deng
authors.affiliation: Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.
authors.forename: Fuyuan
authors.initials: F
authors.lastname: Qiao
authors.affiliation: Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Wu
mesh_terms: D002460:Cell Line; D002465:Cell Movement / Q000235:genetics*; D019175:DNA Methylation / Q000235:genetics*; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D015843:Serpins / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014327:Trophoblasts; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics; D042582:Vascular Endothelial Growth Factor C / Q000235:genetics
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; C085344:SERPIN-B5; D015843:Serpins; C467484:VEGFA protein, human; C467486:VEGFC protein, human; D042461:Vascular Endothelial Growth Factor A; D042582:Vascular Endothelial Growth Factor C; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2