MicroRNA-222调控HUVECc-kit基因表达及对内皮微粒的影响-东南大学学报医学版

MicroRNA-222调控HUVECc-kit基因表达及对内皮微粒的影响

单位：1. 南方医科大学附属深圳市妇幼保健院产科, 广东深圳 518000;
2. 南方医科大学附属深圳市妇幼保健院生殖综合门诊, 广东深圳 518000;
3. 南方医科大学附属深圳市妇幼保健院生殖中心, 广东深圳 518000

分类号：R-33

出版年·卷·期（页码）：2019·38·第二期（280-286）

摘要：

目的：探讨微小RNA （miR）-222及特异性miR-222抑制剂对人脐静脉内皮细胞（HUVEC）c-kit基因的mRNA和蛋白表达、内皮微粒的影响。方法：设计miR-222-3p mimic和miR-222-3p inhibitor及相应的阴性对照（mimic control和inhibitor control），分别转染HUVEC，通过实时荧光定量聚合酶链式反应技术（qRT-PCR）验证转染效率。利用qRT-PCR检测c-kit基因mRNA表达水平；免疫印迹技术分析c-kit蛋白表达水平；CD144标记内皮微粒，流式细胞仪检测miR-222转染对内皮微粒分泌的影响。结果：与mimic control转染组相比，转染miR-222-3p mimic均显著降低c-kit基因mRNA和蛋白表达水平。与inhibitor control转染组相比，转染miR-222-3p inhibitor均显著提高c-kit基因mRNA和蛋白表达水平。流式细胞仪检测显示miR-222-3p表达上调导致内皮微粒分泌增加，miR-222-3p表达下调导致内皮微粒分泌减少。结论：miR-222表达上调抑制c-kit基因mRNA的表达，进而抑制蛋白的表达，miR-222表达上调可能通过引起HUVEC株细胞活化而致内皮微粒分泌增加，进而介导了内皮细胞功能的紊乱。

Objective:To explore the effect of miR-222 and specific miR-222 inhibitor on the expression of mRNA and protein of c-kit gene and its effect on endothelial microparticles in human umbilical vein endothelial cell(HUVEC) line. Methods:MiR-222-3p mimic,mimic negative control, miR-222-3p inhibitor and inhibitor negative control were synthesized and transfected into HUVEC line via transfection reagent Lipofectamine^TM 2000 and the expressions of miR-222 in HUVEC were detected by real time quantitative PCR (qRT-PCR). The expression of mRNA and protein of c-kit gene were detected by qRT-PCR and Western blotting(WB). The influence on endothelial microparticles was detected by flow cytometry. Results:The overexpression of miRNA-222 in HUVEC line strongly downregulated the expression of mRNA and protein of c-kit gene in contrast with the mimic negative control group, in addition induced the secretion of endothelial microparticles. On the contrary, the expression of miR-222 signicantly enhanced the expression of mRNA and protein of c-kit gene in contrast with the inhibitor negative control group, in addition increased the secretion of endothelial microparticles. Conclusion:MiR-222 and c-kit gene and protein expression was negatively correlated. MiR-222 up-regulation may induce the activation of HUVEC, and increase the secretion of endothelial microparticles, which may leads to the dysfunction of endothelial cells.

参考文献：

[1] BUSHATI N,COHEN S M.MicroRNA functions[J].Annu Rev Cell Dev Biol,2007,23:175-205.
[2] LAZAR L,RIGO J.The role of microRNA in pathogenesis of preeclampsia-miRNA network analysis[J].Pregnancy Hypertens,2013,3(2):99.
[3] FRIEDMAN R C,FARH K K,BURGE C B,et al.Most mammalian mRNAs are conserved targets of microRNAs[J].Genome Res,2009,19(1):92-105.
[4] DONEDA L,BULFAMANTE G,GRIMOLDI M G,et al.Localization of fos, jun, kit and SCF mRNA in human placenta throughout gestation using in situ RT-PCR[J].Early Pregnancy,1997,3(4):265-271.
[5] 秦薇,高洁.miR-221/miR-222与c-Kit在重度子痫前期的表达及意义[J].赣南医学院学报,2015,35(6):887-890.
[6] 樊晟,陈玉梅,李六兰,等.妊娠期糖尿病脐血miR-222表达水平及其与子代出生体重的相关性分析[J].东南大学学报(医学版),2014,33(6):706-709.
[7] SITRAS V,PAULSSEN R H,GRONAAS H,et al.Differential placental gene expression in severe preeclampsia[J].Placenta,2009,30(5):424-433.
[8] FOUNDS S A,CONLEY Y P,LYONS-WEILER J F,et al.Altered global gene expression in first trimester placentas of women destined to develop preeclampsia[J].Placenta,2009,30(1):15-24.
[9] LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[J]. Methods,2001,25(4):402-408.
[10] 李朝曦,苏放明.子痫前期预测的最新研究进展[J].现代医学,2012,40(1):118-121.
[11] MURPHY M S,CASSELMAN R C,TAYADE C, et al.Differential expression of plasma microRNA in preeclamptic patients at delivery and 1 year postpartum[J].Am J Obstet Gynecol,2015,213(3):367.
[12] LUO S S,ISHIBASHI O,ISHIKAWA G,et al.Human villous trophoblasts express and secrete placenta-specific microRNAs intomaternal circulation via exosomes[J].Biol Reprod,2009,81(4):717-729.
[13] MORALES PRIETO D M,MARKERT U R.MicroRNAs in pregnancy[J].Journal of Reproductive Immunology,2011,88:106-111.
[14] DENTELLI P,ROSSO A,ORSO F,et al.MicroRNA-222 controls neovascularization by regulating signal transducer and activator of transcription 5A expression[J].Arterioscler Thromb Vasc Biol,2010,30(8):1562-1568.
[15] AHMED A,LACSON A,GILBERT-BARNESS E.Immunohistochemical expression of endothelial nitric oxide synthase and C-kit in the placenta of complete hydatidiform mole[J].Fetal Pediatr Pathol,2005,24(3):141-147.
[16] ZHONG H L,LU X Z,CHEN X M,et al.Relationship between stem cell factor/c-kit expression in peripheral blood and blood pressure[J].J Hum Hypertens,2010,24(3):220-225.
[17] GITS C M,van KUIJK P F,JONKERS M B,et al.MiR-17-92 and miR-221/222 cluster members target KIT and ETV1 in human gastrointestinal stromal tumours[J].Br J Cancer,2013,109(6):1625-1635.
[18] KE J,ZHAO Z,HONG S H,et al.Role of microRNA221 in regulating normal mammary epithelial hierarchy and breast cancer stem-like cells[J].Oncotarget,2015,6(6):3709-3721.
[19] POLISENO L,TUCCOLI A,MARIANI L,et al.MicroRNAs modulate the angiogenic properties of HUVECs[J].Blood,2006,108(9):3068-3071.
[20] BRODSKY S V,ZHANG F,NASJLETTI A,et al.Endothelium-derived microparticles impair endothelial function in vitro[J].Am J Physiol Heart Cire Physiol,2004,286(5):H1910-1915.
[21] JANSEN F,YANG X,BAUMANN K,et al.Endothelial microparticles reduce ICAM-1 expression in a microRNA-222-dependent mechanism[J].J Cell Mol Med,2015,19(9):2202-2214.

服务与反馈：

【文章下载】【发表评论】【查看评论】【加入收藏】

提示：您还未登录，请登录！点此登录