缺氧通过微小RNA-155和cyclin D1抑制滋养细胞迁移  

Hypoxia-induced microRNA-155 inhibits migration of human-trophoblast-derived HTR-8/SVneo cells by targeting eyelin D1

作　　者：薛平平[1] 李玉静[2] 戴毅敏[2] 邱智华[2] 沈莉[1] 刁振宇[2] 颜桂军[2] 胡娅莉[1] 

机构地区：[1]南京医科大学鼓楼临床医学院妇产科,210029 [2]南京大学医学院附属鼓楼医院妇产科

出　　处：《中华围产医学杂志》2015年第3期214-221,共8页Chinese Journal of Perinatal Medicine

基　　金：国家自然科学基金（81370724,81070508）;江苏省2013年度普通高校研究生科研创新计划项目（CXLX13-568）

摘　　要：目的探讨缺氧对人绒毛外滋养细胞微小RNA（microRNA，miRNA）-155表达的影响及其对滋养细胞迁移的影响。方法（1）采用100μmol／L氯化钴（cobaltchloride，CoCI2）诱导HTR-8／SVneo细胞缺氧，实时定量聚合酶链反应技术检测miRNA-155的表达，蛋白质印迹技术检测JunB和FosB蛋白的表达，划痕实验评估细胞迁移能力。（2）采用SP600125和PDTC分别抑制激活蛋白-1（activeprotein-1，AP-1）和／或核因子（nuclearfactor）-KB通路，实时定量聚合酶链反应技术检测miRNA-155的表达变化。（3）HTR-8／SVneo细胞瞬时转染pEGFP—miRNA—155、pEGFP—C1和pGL3-pro～cyclinD13-UTR，荧光素酶报告基因系统检测miRNA—155对cyclinD13＇非编码区的调控。（4）HTR-8／SVneo细胞瞬时转染pEGFP—miRNA-155和／或pFLAG—CMV—cyclinD1以过表达miRNA-155和／或cyclinD1，划痕实验评估细胞迁移能力的改变。采用两独立样本t检验，方差分析及LSD检验进行统计学分析。结果（1）100μmol／LCoCl2培养HTR-8／SVneo细胞24和48h，miRNA-155的相对表达量分别是0h的（1．40±0．28）倍（t=3．302，P=0．030）和（1．74±0．14）倍（t=8．578，P=0．001），随CoCl，（100μmol／L）作用于HTR-8／SVneo细胞的时间延长，JunB和FosB蛋白表达均升高。细胞迁移率在培养12、24和48h较0μmol／LCoC12时下降，尤以48h降低明显[（52．98±3．77）％与（64．68±3．92）％，t=5．259，P=0．000]。（2）抑制AP-1亚组、抑制NF—KB亚组及同时抑制AP—1和NF—KB亚组miRNA-155的相对表达量分别降低至无抑制情况下的（50．45±3．53）％、（47，18±2．14）％和（66．79±3．92）％（t值分别为3．630、4．100和3．392，P值均〈0．05）。（3）过表达miRNA-155亚组的cyclinD13’非编码区荧光素酶活性较无过表达时降低（t=46．682，P=0．000）。（4）过表达miRNA-155亚组的HTR-8／SVneo细胞迁移率较无过表达时明显降低[48h，（Objective To investigate the regulation of microRNA （miRNA）-155 expression under hypoxia and its effects on migration oftrophoblast cells. Method （1） Cobalt chloride （COC12） （100 μmol/L） was used to induce hypoxia in cultured human-trophoblast-derived HTR-8/SVneo cells, quantitative real-time polymerase chain reaction （PCR） was used to detect the expression of miRNA-155, JunB and FosB protein were then evaluated using Western blot, and wound healing assays were performed to assess cell migration. （2） SP600125 and/or PDTC were added to inhibit activation of the active protein-1 （AP-1） and nuclear factor- κ B （NF- κ B） pathways, and miRNA-155 was tested by quantitative real-time PCR. （3） HTR-8/SVneo cells were co-transfected with plasmids containing pEGFP-miRNA- 155/pEGFP-C 1 and pGL3-pro-cyclin D 1 3＇UTR, and luciferase reporter assays were used to assess the regulatoin of cyclin D1 ＇3 untranslated region （3＇UTR） by miRNA-155. （4） HTR-8/SVneo cells were co-transfected with plasmids containing pEGFP-miRNA- 155/pEGFP-C 1 and pFLAG-CMV-cyclin D 1/pFLAG-CMV-2 to induce overexpression of miRNA-155 and/or cyclin D1, and wound healing assays were used to assess cell migration. The two independent-samples t test, one-way analysis of variance and LSD test were used for statistical analysis. Results （1） Compared to cells cultured without CoCl：, hypoxia, induced by CoCl2 （100 μ mol/L） for 24 and 48 h, induced enhanced expression of miRNA-155 [（1.40±0.28） fold and （1.744-0.14） fold, t=3.302 and 8.578, P=0.030 and 0.001, respectively], JunB and FosB protein were upregulated by hypoxia induced by 100 μmol/L CoCl2. However, migration rate decreased at 12, 24 and 48 b, especially at 48 h [（52.98±3.77）% vs （64.68±3.92）%, t=5.259, P=0.000].（2） In the AP-1 inhibited subgroup, NF- κ B inhibited subgroup and AP-1＋NF- κ B inhibited subgroup, miRNA- 155 was downregulated to （50.45 ± 3.53）%, （47.18 ± 2.14）% and （66.79_ 3.92）% of the

关 键 词：缺氧 微RNAS 细胞周期蛋白D1 滋养层 细胞迁移抑制 先兆子痫 

分 类 号：R714.2[医药卫生—妇产科学]