| Part One.The effects of miR-210 on human trophoblast cell line HTR-8/SVneo function ObjectiveWe aimed to investigate the changes in cell proliferation、apoptosis、migration、invasion and tube-like formation after regulating miR-210 expression in HTR-8/SVneo cells.Methods1.In-situ hybridization was applied to localize the expression of miR-210 in placental villi and maternal decidua of early gestation.2.HTR-8/SVneo cells was transfected with miR-210 mimic and miR-210 mimic NC,and thus divided into overexpression group and negative control group.qRT-PCR was performed to detect the expression of miR-210 in these two groups.3.HTR-8/SVneo cells was transfected with miR-210 inhibitor and miR-210 inhibitor NC,and thus divided into down-regulation group and negative control group.qRT-PCR was performed to detect the expression of miR-210 in these two groups.4.CCK-8 assay was performed to evaluate cell proliferation ability.5.Flow cytometry was performed to evaluate cell apoptosis ability.6.Transwell assays were conducted to detect cell migration and invasion capacities.7.Network formation assay was performed to evaluated cell angiogenesis ability.8.qRT-PCR and Western Blot were performed to detect the expression of MMP2、Bcl-2、BAX、VEGF-A、PIGF in transfected HTR-8/SVneo cells from RNA and protein level.Results1.miR-210 was expressed in both trophoblasts in villi and EVTs in maternal decidual,and its expression level in decidual EVTs is lower than villous trophoblastcells.2.Compared with miR-210 mimic NC group,the expression of miR-210 was significantly increased in overexpression group.Increased apoptosis level,decreased cellular activity,migration and invasion capacity,and tube formation ability can be observed in the miR-210 mimic group;3..Compared with miR-210 inhibitor NC group,the expression of miR-210 was significantly decreased in down-regulation group.Decreased apoptosis level,increased cellular activity,migration and invasion capacity,and tube formation ability can be observed in the miR-210 inhibitor group;4.Compared with miR-210 mimic group,the m RNA level and protein level of MMP2、Bcl-2、VEGF-A、PIGF in overexpression group were obviously decreased,while the BAX expression was increased;5.Compared with miR-210 inhibitor group,the m RNA level and protein level of MMP2、Bcl-2、VEGF-A、PIGF in overexpression group were obviously up-regulated,while the BAX expression was decreased;ConclusionRegulation of miR-210 expression could change the proliferation、 apoptosis、migration、 invasion and tube formation ability of HTR-8/SVneo cells,suggesting that miR-210 may have an effect on EVTs cells biological function.Part two.miR-210 regulated the NOTCH1 expression to modulate trophoblast migration and invasion ObjectiveThis study is conducted to investigate the role of miR-210 and its relationship with NOTCH1 in trophoblast cells.Methods1.Both m RNA and protein expression levels of NOTCH1 were measured in preeclamptic placenta(n = 17)and normal placenta(n = 17);The m RNA expression level of miR-210 was also measured.2.The expression and location of NOTCH1 in first-trimester villi and decidua were shown via immunohistochemistry.3.Trophoblast cell line HTR-8/SVneo was used to explore the effect of miR-210 on NOTCH1 expression and trophoblast bioactivity(including migration and invasion)by gain-and loss-of function studies.Results1.miR-210 expression was increased in preeclamptic placenta compared with normal placenta,while the NOTCH1 expression was decreased.2.NOTCH1 was expressed in both trophoblasts in villi and EVTs in maternal decidual,and its expression level in decidual EVTs is higher than villous trophoblast cells.3.Down-regulation of miR-210 could promote NOTCH1 expression and increased HTR-8/SVneo cells migration and invasion.ConclusionmiR-210 could attenuate NOTCH1 expression and further inhibited trophoblasts migration and invasion,which may be an important mechanism of spiral artery remodeling disorder. |
