Study On The Relationship Between The High Expression Of Siglec6 And The Pathogenesis Of Preeclampsia

Background&ObjectivesPreeclampsia(PE),one of the most common diseases during pregnancy,complicates 2-8%of pregnancies globally and is a major cause of maternal as well as perinatal morbidity and mortality.It has lasting effects on both mothers and children,resulting in increased susceptibility to hypertension and chronic kidney disease with increasing lifelong risk in both individuals.The pathogenesis of PE is currently unclear,and the only known cure for PE is delivery of the placenta,which often results in premature delivery.Exploring the pathogenesis of PE and finding new treatments are necessary for the complications associated with PE that seriously increase health and financial burdens,especially in developing countries.Siglec6 is a member of the sialic acid-binding immunoglobulin-like lectin family.It is highly expressed in human placental trophoblast cells and is involved in regulating the function of trophoblast cells.Previous studies have shown that Siglec6 is abnormally expressed in the PE placenta,but the role of Siglec6 in the PE pathogenesis is unclear.The purposes of this study are to clarify the expression of Siglec6 in the PE placenta and to explore the relationship between Siglec6 and the pathogenesis of PE.Methods1.Detect the expression of Siglec6 in the placenta:All placental samples were collected at the Shenzhen Maternity&Child Healthcare Hospital between June 2019 and December 2019(approved by the Medical Ethics Committee of Shenzhen Maternity&Child Healthcare Hospital and obtained by the pregnant woman’s informed consent),including 5 normal placentas and 5 preeclampsia placentas.The expression levels of Siglec6 in normal and PE placentas were compared by Q-PCR and Western blot experiments.Finally,the expression of Siglec6 was localized by immunohistochemistry experiments.2.The upregulation of Siglec6 in trophoblast cells induced by hypoxia:CoCl2 and DMOG were added to the culture medium of HTR8/SVneo and BeWo cells.Cellular immunofluorescence and Western blot experiments were used to demonstrate the induction of hypoxia-inducible factor-1α(HIF-1α),and then Q-PCR and Western blot experiments were used to detect the expression of Siglec6.3.The effect of Siglec6 overexpression on HTR8/SVneo cell functions:Lentiviral overexpression vector was used to construct Siglec6 overexpressing HTR8/SVneo cells.CCK-8 was used to detect cell proliferation,and Q-PCR experiments were used to detect the expressions of genes related to cell growth.Migration and invasion capabilities were detected by Transwell experiments.4.The effect of elevated trophoblastic Siglec6 on the function of human umbilical vein endothelial cells(HUVEC):The conditioned medium derived from HTR8/SVneo cells were collected to treat HUVEC.Then,the proliferation,migration,invasion,and tube formation of HUVEC were detected.5.The mechanism of HUVEC function impairment induced by elevated trophoblastic Siglec6:Transcriptome sequencing of HTR8/SVneo cells found that the expression of Wnt6 was down-regulated,and the content of Wnt6 in the conditioned medium was detected by ELISA.Recombinant human Wnt6(rhWnt6)was used to stimulate HUVECs in vitro and rhWnt6 was added to the conditioned medium of HTR8/SVneo cells for rescue experiments.Finally,the expression of Wnt/β-catenin signaling was detected by Western blot experiments in HUVEC.Results1.Compared with normal pregnancies placenta,the mRNA and protein expression levels of Siglec6 in the PE placenta were significantly increased.Besides Siglec6 was primarily expressed in trophoblast cells,including those in the syncytiotrophoblast(STB)and extravillous trophoblast(EVT).2.Both CoCl2 and DMOG induced HIF-1α accumulation in the nucleus.Moreover,both reagents regulated the expression of Siglec6 in HTR8/SVneo and BeWo cells.3.Siglec6 overexpression had no effect on HTR8/SVneo cell proliferation,migration,and invasion.4.Conditioned medium(CM)derived from Siglec6-overexpressing HTR8/SVneo cells(Siglec6-OE-CM)impaired HUVEC proliferation,migration,invasion,and tube formation.5.Overexpression of Siglec6 altered the expression levels of many genes in HTR8/SVneo cells,including down-regulated Wnt6.6.rhWnt6 promoted the proliferation,migration,invasion,and tube formation of HUVECs and rescued the compromised functions of HUVECs caused by Siglec6-OE-CM.Moreover,rhWnt6 mediated the Wnt/β-catenin signaling pathway to regulate HUVEC functions.ConclusionSiglec6 is upregulated in trophoblast cells of the PE placenta,which may be caused by hypoxia.The overexpression of Siglec6 inhibits the secretion of Wnt6 by trophoblast cells,which further contributes to the impairment of VEC function in a paracrine fashion.These findings provide new insights into the pathogenesis and treatment of PE.