miR-409-3p靶向CXCL9调控滋养层细胞的增殖、迁移和侵袭

miR-409-3p regulates the proliferation, migration and invasion of trophoblast cells by targeting CXCL9

目的探讨微小RNA-409-3p(miR-409-3p)对滋养层细胞增殖、迁移和侵袭影响及作用机制。方法实时荧光定量PCR(RT-qPCR)检测正常妊娠胎盘组织和子痫前期胎盘组织中miR-409-3p和干扰素伽玛诱导的单核细胞因子(CXCL9)mRNA表达水平,蛋白印迹(Western blot)法检测CXCL9蛋白表达水平。转染miR-409-3p模拟物或CXCL9小干扰RNA至滋养层细胞HTR8/SVneo,构建miR-409-3p过表达或CXCL9表达抑制的HTR8/SVneo细胞,四甲基噻唑蓝染色法(MTT)检测细胞增殖,Transwell检测细胞迁移和侵袭,Western blot检测细胞周期蛋白D1(CyclinD1)、p21、基质金属蛋白酶2(MMP-2)和MMP-9蛋白表达水平。双荧光素酶报告基因实验验证miR-409-3p与CXCL9调控关系。结果与正常妊娠胎盘组织比较,子痫前期胎盘组织中miR-409-3p水平升高(P<0.05),CXCL9 mRNA和蛋白水平降低(P<0.05)。miR-409-3p过表达或干扰CXCL9表达后,HTR8/SVneo细胞培养48 h和72 h后OD值、迁移和侵袭数、CyclinD1、MMP-2和MMP-9蛋白表达水平降低(P<0.05),p21蛋白表达水平升高(P<0.05)。miR-409-3p在HTR8/SVneo细胞中靶向负调控CXCL9表达。CXCL9过表达逆转了miR-409-3p过表达对HTR8/SVneo细胞增殖、迁移和侵袭的影响。结论 miR-409-3p抑制HTR8/SVneo细胞的增殖、迁移和侵袭与下调CXCL9表达有关。

This study was designed to investigate the effects of miR-409-3 p on the proliferation, migration and invasion of trophoblast cells and its mechanism. Real-time quantitative PCR(RT-qPCR) was used to detect the expression of miR-409-3 p and CXCL9 mRNA in normal placenta and preeclamptic placenta, while Western blot was used to detect the expression of CXCL9 protein. miR-409-3 p mimic or CXCL9 small interfering RNA was transfected into trophoblast cell HTR8/SVneo to construct HTR8/SVneo cells with miR-409-3 p overexpression or CXCL9 expression inhibition. MTT assay was used to detect the cell proliferation, Transwell was used to detect the cell migration and invasion, while Western blot was used to detect the expression of Cyclin D1, p21, MMP-2 and MMP-9 proteins. Compared with normal placenta tissue, the level of miR-409-3 p in placental tissue of preeclampsia was increased(P<0.05), and the levels of CXCL9 mRNA and protein were decreased(P<0.05). After miR-409-3 p overexpression or interference with CXCL9 expression, the OD value at 48 h and 72 h, the migration and invasion number and the expression levels of Cyclin D1, MMP-2 and MMP-9 protein were decreased(P<0.05),while the expression level of p21 protein was increased(P<0.05). miR-409-3 p regulated CXCL9 expression negatively in HTR8/SVneo cells;overexpression of CXCL9 reversed the effects of miR-409-3 p overexpression on the proliferation, migration and invasion of HTR8/SVneo cells. Taken together, miR-409-3 p inhibits the proliferation, migration and invasion of HTR8/SVneo cells and the mechanism is associated with down-regulation of CXCL9 expression.