缺氧通过微小RNA-155和cyclin D1抑制滋养细胞迁移
결양통과미소RNA-155화cyclin D1억제자양세포천이
Hypoxia-induced microRNA-155 inhibits migration of human-trophoblast-derived HTR-8/SVneo cells by targeting cyclin D1
  • 万方数据
    中华围产医学杂志 중화위산의학잡지
    CHINESE JOURNAL OF PERINATAL MEDICINE
    2015年 3期 214-221 ,共8页

    薛平平%李玉静%戴毅敏%邱智华%沈莉%刁振宇%颜桂军%胡娅莉

    설평평%리옥정%대의민%구지화%침리%조진우%안계군%호아리

    缺氧%微RNAs%细胞周期蛋白D1%滋养层%细胞迁移抑制%先兆子痫 缺氧%微RNAs%細胞週期蛋白D1%滋養層%細胞遷移抑製%先兆子癇
    결양%미RNAs%세포주기단백D1%자양층%세포천이억제%선조자간
    Anoxia%MicroRNAs%Cyclin D1%Trophoblasts%Cell migration inhibition%Pre-eclampsia
    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(cobalt chloride,CoCl2)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(active protein-1,AP-1)和/或核因子(nuclear factor)-κ B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-cyclin D1 3'UTR,荧光素酶报告基因系统检测miRNA-155对cyclin D1 3'非编码区的调控.(4)HTR-8/SVneo细胞瞬时转染pEGFP-miRNA-155和/或pFLAG-CMV-cyclin D1以过表达miRNA-155和/或cyclin D1,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) 100 μmol/L CoCl2培养HTR-8/SVneo细胞24和48 h,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CoC12(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48 h较0μmol/L CoC12时下降,尤以48 h降低明显[(52.98±3.77)％与(64.68±3.92)％,t=5.259,P=0.00 0].(2)抑制AP-1亚组、抑制NF-κB亚组及同时抑制AP-1和NF-κB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cyclin D1 3'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[48 h,(33.31±6.19)％与(47.20±2.82)％,LSD检验,P＜0.05],而同时过表达miRNA-155和cyclin D1亚组的HTR-8/SVneo细胞迁移率较单纯过表达miRNA-155亚组明显升高[48 h,(43.04±1.44)％与(33.31±6.19)％,LSD检验,P=0.002]. 结论 缺氧通过激活HTR-8/SVneo细胞中AP-1和NF-κB通路而引起miRNA-155的表达升高,miRNA-155表达升高可以下调细胞周期蛋白cyclin D1,进而抑制滋养细胞迁移.
    목적 탐토결양대인융모외자양세포미소RNA(microRNA,miRNA)-155표체적영향급기대자양세포천이적영향. 방법 (1)채용100μmol/L록화고(cobalt chloride,CoCl2)유도HTR-8/SVneo세포결양,실시정량취합매련반응기술검측miRNA-155적표체,단백질인적기술검측JunB화FosB단백적표체,화흔실험평고세포천이능력.(2)채용SP600125화PDTC분별억제격활단백-1(active protein-1,AP-1)화/혹핵인자(nuclear factor)-κ B통로,실시정량취합매련반응기술검측miRNA-155적표체변화.(3)HTR-8/SVneo세포순시전염pEGFP-miRNA-155、pEGFP-C1화pGL3-pro-cyclin D1 3'UTR,형광소매보고기인계통검측miRNA-155대cyclin D1 3'비편마구적조공.(4)HTR-8/SVneo세포순시전염pEGFP-miRNA-155화/혹pFLAG-CMV-cyclin D1이과표체miRNA-155화/혹cyclin D1,화흔실험평고세포천이능력적개변.채용량독립양본t검험,방차분석급LSD검험진행통계학분석. 결과 (1) 100 μmol/L CoCl2배양HTR-8/SVneo세포24화48 h,miRNA-155적상대표체량분별시0h적(1.40±0.28)배(t=3.302,P＝0.030)화(1.74±0.14)배(t=8.578,P=0.001),수CoC12(100μmol/L)작용우HTR-8/SVneo세포적시간연장,JunB화FosB단백표체균승고.세포천이솔재배양12、24화48 h교0μmol/L CoC12시하강,우이48 h강저명현[(52.98±3.77)％여(64.68±3.92)％,t=5.259,P=0.00 0].(2)억제AP-1아조、억제NF-κB아조급동시억제AP-1화NF-κB아조miRNA-155적상대표체량분별강저지무억제정황하적(50.45±3.53)％、(47.18±2.14)％화(66.79±3.92)％(t치분별위3.630、4.100화3.392,P치균＜0.05).(3)과표체miRNA-155아조적cyclin D1 3'비편마구형광소매활성교무과표체시강저(t=46.682,P=0.000).(4)과표체miRNA-155아조적HTR-8/SVneo세포천이솔교무과표체시명현강저[48 h,(33.31±6.19)％여(47.20±2.82)％,LSD검험,P＜0.05],이동시과표체miRNA-155화cyclin D1아조적HTR-8/SVneo세포천이솔교단순과표체miRNA-155아조명현승고[48 h,(43.04±1.44)％여(33.31±6.19)％,LSD검험,P=0.002]. 결론 결양통과격활HTR-8/SVneo세포중AP-1화NF-κB통로이인기miRNA-155적표체승고,miRNA-155표체승고가이하조세포주기단백cyclin D1,진이억제자양세포천이.
    Objective To investigate the regulation of microRNA (miRNA)-155 expression under hypoxia and its effects on migration oftrophoblast cells.Method (1) Cobalt chloride (CoCl2) (100 μ mol/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-C1 and pGL3-pro-cyclin D1 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 CoC12,hypoxia,induced by CoC12 (100 μ mol/L) for 24 and 48 h,induced enhanced expression of miRNA-155 [(1.40±0.28) fold and (1.74±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 μ mol/L CoC12.However,migration rate decreased at 12,24 and 48 h,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 noninhibited subgroup (t was 3.630,4.100 and 3.392,all P ＜ 0.05,respectively).(3)The luoiferase activity of cyclin D1 YUTR was significatly decreased in the overexpression miRNA-155 subgroup than in the normal expression miRNA-155 subgroup (t=46.682,P=0.000).(4) The migration rates of HTR-8/SVneo cells in the overexpression miRNA-155 subgroup were lower than in the normal-expression miRNA-155 subgroup [at 48 h,(33.31 ± 6.19)％vs (47.20±2.82)％,LSD test,P=0.002,respectively],and in the overexpression miRNA-155 + cyclin D1 subgroup was higher than the overexpression miRNA-155 subgroup [at 48 h,(43.04 ± 1.44)％ vs (33.31 ± 6.19)％,LSD test,P=0.002,respectively].Conclusions Hypoxia induces the expression ofmiRNA-155 via activation of the AP-1 and NF-κ B pathways.Overexpression of miRNA-155 inhibits trophoblast migration by downregulating cyclin D1.
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