摘要
目的目前虽已发现一些在胚胎着床过程中起重要作用的分子,但微小RNA(microRNA,miRNA)在蜕膜化过程中作用的研究报道较少,文中调查孕鼠围着床期子宫组织中miR-181a的表达规律,并探讨miR-181a对人子宫内膜间质细胞(human endometrial stromal cell,hESC)体外诱导蜕膜化过程中蜕膜化标志基因蜕膜化催乳素(decidual prolactin,dPRL)表达的调控作用。方法收集怀孕0～6.5 d孕鼠子宫组织,采用TRIZOL法提取总RNA,通过实时定量PCR检测子宫组织中miR-181a的表达。制备Ad-miR-181a重组腺病毒,并感染hESC,24 h后采用0.5 mmol/L 8-Br-cAMP和1μmol/L Medroxyproges-terone(MPA)联合进行体外蜕膜化诱导培养;通过化学发光免疫法和实时定量PCR分别检测细胞培养液上清中dPRL的浓度与间质细胞中催乳素(prolactin,PRL)mRNA的表达;同时采用荧光素酶报告基因检测miR-181a对hESC中dPRL(-332/+65)启动子的调控作用。结果早孕小鼠子宫组织中miR-181a的表达高于非妊娠小鼠子宫,且随着妊娠天数的增加呈逐渐上升的趋势,在小鼠着床"窗口期"即孕4.5 d达到高峰(2.60±0.15倍,P<0.01,n=5),孕5.5 d子宫miR-181a的表达开始平稳下降;成功获得滴度为5.19×1010ifu/ml的miR-181a腺病毒,该腺病毒介导的miR-181a过表达显著增加hESC蜕膜化标志基因dPRL mRNA表达水平,增高超过8倍(P<0.01);在8-Br-cAMP和MPA联合诱导hESC体外蜕膜化时,高表达miR-181a可明显增加8-Br-cAMP联合MPA诱导的PRL mRNA表达水平与PRL蛋白分泌(P<0.01)。荧光素酶报告基因进一步证实miR-181a可以激活dPRL(-332/+65)启动子的活性达到2倍(P<0.05)。结论 miR-181a参与8-Br-cAMP和MPA诱导子宫内膜间质细胞蜕膜化过程中PRL的表达调控。
Objective Though some molecules have been found to play important parts in embryo implantation, few reports are seen on the roles of microRNAs （miRNAs） in decidualization. This study aimed to investigate the expression of miR-181a in the mouse uterus during peri-implantation of embryos and the role of miR-181a in regulating the expression of decidual prolactin （dPRL） inhuman endometrial stromal cells （hESCs）. Methods The expression of miR-181a in the uterus of pregnant mice after 0 -6.5 d of copulation was detected by quantitative real-time PCR. The hESCs were transfected with recombinant adenovirus of Ad-miR-181a and 24 h later cultured by in vitro decidual induction with 0.5 mmol/L of 8-Br-cAMP and 1 p.mol/L of medroxyprogesterone （MPA）. The con- centration of dPRL in the culture supernatant and the mRNA expres- sion of prolactin （PRL） in the stromal cells were detected by quanti- tative real-time PCR, and the role of miR-181a in regulating the dPRL（-332/＋ 65 ） promoter in the hESCs determined by lucifera^e assay. Results The expression of miR-181a was higher in the uterus of the early pregnant mice than in that of the non-pregnant ones, and exhibited a rising trend with the increased time of pregnan-cy, reaching the maximum at 4.5 d [ （2.60±0.15） times, P 〈 O. 01, n = 5 ] and beginning to decrease steadily at 5.5 d. We suc- cessfully obtained 5.19±10^10 ifu/ml adenovirus of miR-181 a. Adenovirus-mediated overexpression of miR-181 a in hESCs markedly in- creased the mRNA expression dPRL by 9 times （P 〈 O. 01 ） and enhanced dPRL promoter （ dPRL/- 332Luc） activity and dPRL se- cretion （P 〈 O. 01 ）. Furthermore, the overexpression of miR-181 a significantly promoted the induction of PRL mRNA expression and protein secretion by 8Br-cAMP and MPA （P 〈0.01 ）. Conclusion miR-181a is involved in the regulation of the dPRL expression during the decidualization of human endomentiral stromal ceils induced by 8Br-cAMP and MPA.
出处
《医学研究生学报》
CAS
北大核心
2012年第2期129-134,共6页
Journal of Medical Postgraduates
基金
国家重点基础研究发展计划(2010CB945104)
国家自然科学基金(30900727,81070492,81070508)
江苏省自然科学基金(BK2009038)
南京市卫生局资助项目(QYK09172)
