CN114438187B - 一种诊断早期流产的miRNA标志物及其应用 - Google Patents
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Abstract
本发明公开一种用于早期流产诊断的新的生物标志物,具体涉及hsa‑miR‑29a‑3p在诊断、预测和/或监测早期流产的方法及应用。本发明首次提出hsa‑miR‑29a‑3p作为早期妊娠胚胎植入及不良妊娠结局的标志物,然后利用体外子宫内膜基质细胞蜕膜化模型证实hsa‑miR‑29a‑3p对蜕膜化的调控作用。基于该标志物的时空表达特异性将具有广阔的临床诊断和预测应用前景。本发明所述hsa‑miR‑29a‑3p在不同妊娠时期及在早期流产蜕膜组织中的的特异性表达,能有效的预测和诊断妊娠早期流产。
Description
所属技术领域
本发明属于生物检测与诊断领域,具体涉及一种妊娠早期流产诊断用的miRNA生物标志物及其应用。
背景技术
流产是一个普遍的问题,会影响39岁以下25%怀孕妇女。据统计流产影响大约三分之一的妊娠妇女,并且最常发生在孕中期和早孕期。近年来,早期流产发病逐年增长,尽管随着早期干预方法及药物治疗的发展,对早期流产的预防取得一定的效果,然而,早期流产预测及诊断方法的缺乏使得该妊娠疾病难以有效防控。
母胎间正常交流是维持成功和健康怀孕的基础。子宫内膜***(ESCs)的不断增殖与分化,在妊娠初期达到蜕膜化状态。ESCs蜕膜功能失调阻碍了滋养层细胞侵入子宫内膜,可导致包括不育,早期流产,宫内发育迟缓和先兆子痫等妊娠相关疾病。有研究表明,HOXA10、IL-6可调节ESCs细胞增殖和蜕膜化。此外,类固醇激素和粘附分子也参与ESCs细胞增殖和蜕膜化的调节,并进一步影响胚胎植入和早期妊娠。但是,导致蜕膜化缺陷的分子机制及其在早期流产中发挥的作用目前仍然不清楚。
微小RNA(miRNA)是一类小的非编码RNA,能通过与靶向mRNA的3'-UTR结合来负调控基因的表达。研究发现,在蜕膜化过程和胚胎植入的窗口期中,miRNA在子宫内膜中存在差异表达。已证实miR-98通过靶向大鼠子宫中的Bcl-xl来调控ESCs的增殖与凋亡。最新一项研究显示,流产患者的蜕膜组织中miR-24下调,并通过调节ESCs的增殖和凋亡影响妊娠结局。但是,仍有许多未知的miRNA参与调控ESCs的蜕膜化过程。据报道,miR-29a能诱导滋养层细胞凋亡并调节滋养层细胞在流产和胎盘增生中的功能。另一项研究表明,miR-29a通过靶向***中的芳香化酶表达和***生物合成促进颗粒细胞增殖。此外,已证实miR-29a-5p通过靶向TPX2抑制子宫内膜癌细胞增殖。值得注意的是,miR-29a能通过靶向凋亡因子抑制ESCs的凋亡并参与调控在胚胎植入过程。然而,miR-29a影响ESCs的增殖和蜕膜化的作用及其作为早期流产分子标志物的潜在应用仍有待阐明。
目前,早期流产没有有效的分子标志物,由于现有治疗与干预技术的仍然不足,接受流产后的治疗不尽人意,本发明的一种包含miR-29a调控ESCs的增殖和蜕膜化作用及评估了不同妊娠时期及早期流产中miR-29a的表达水平,并评估了miR-29a应用于早期流产的诊断价值。
发明内容
本发明针对现有技术的不足,提供一种一种妊娠早期流产诊断用的miRNA生物标志物及其应用。
在本发明中,所述miRNA生物标志物为hsa-miR-29a-3p(MIMAT0000086)。
所述hsa-miR-29a-3p的序列为:UAGCACCAUCUGAAAUCGGUUA。
优选的,所述hsa-miR-29a-3p为蜕膜组织miRNA生物标志物。
本发明的蜕膜组织miRNA生物标志物,可用于妊娠早期流产的检测,也可用于不同妊娠周期的监测。
本发明提供所述蜕膜组织miRNA生物标志物在筛选或制备早期流产诊断药物中的用途。
本发明提供一组早期流产诊断所用miRNA引物,所述miRNA引物为hsa-miR-29a-3p引物。
所述hsa-miR-29a-3p引物序列为:miR-29a-3p正向引物:5’-TGCGCTAGCACCATCTGAAATC-3’和反向引物:5’-CCAGTGCAGGGTCCGAGGTATT-3’。
优选的,所述miRNA标志物、引物及组合在治疗早期流产的应用。
本发明的目的是按以下技术方案来实现的:
本发明通过***的研究不同妊娠周期及流产患者蜕膜组织miR-29a表达差异。发现分泌期子宫内膜相比增生期子宫内膜组织miR-29a显著上调。与正常早期妊娠相比,早期流产子宫内膜蜕膜组织miR-29a的表达显著降低。与临床组织样本一致,细胞实验证明本发明所述miR-29a在ESCs蜕膜化模型中的表达上调。而miR-29a的下调显着抑制了蜕膜化ESCs的细胞增殖以及蜕膜化标志物PRL和IGFBP1的表达水平。本方法可以有效的预测不同妊娠周期及早期流产。研究结果为早期流产的预测和诊断提供了依据,同时为早期流产分子治疗在临床上的应用奠定了基础。
附图说明
图1为不同时期子宫内膜或蜕膜组织miR-29a的表达差异。
图2为蜕膜化的ESCs中miR-29a表达变化。
图3为miR-29a下调对蜕膜化ESCs的细胞增殖影响。
图4A-B为miR-29a下调对蜕膜化ESCs的蜕膜标志物PRL和IGFBP1的蛋白水平及相对定量的影响。
具体实施案例
为使本发明更加容易理解,下面将进一步阐述本发明的具体实施例。
实施例1:qRT-PCR检测不同妊娠周期子宫内膜、早期流产患者蜕膜组织miR-29a的表达水平
(1)RNA提取:
用手术剪剪取部分组织置于含1mL的Trizol的匀浆管中,使用电动匀浆机充分研磨后(冰上操作),温育5min,12000rpm,离心10min。吸上清于新的1.5mL离心管中,加入200μL的氯仿,摇匀,室温静置2min,4℃,12000rpm,离心10min。吸取上清于新的1.5mL离心管中,加入600μL的异丙醇,混合均匀,室温静置15min,4℃,12000rpm,离心15min,弃上清。加入1mL75%的无水乙醇(750μL无水乙醇和250μLDEPC水)漂洗沉淀,4℃,12000rpm,离心5min,弃上清。加入1mL无水乙醇,漂洗沉淀,4℃,12000rpm,离心5min,弃上清,室温干燥10min。加入40μL的DEPC水溶解RNA,置于-80℃冰箱保存备用。
(2)逆转录反应:
逆转录反应条件:25℃5min,50℃15min,85℃5min,4℃10min;反应体系见表1。
表1逆转录反应体系
(3)qPCR反应:
cDNA做7倍稀释,在如下反应条件下进行反应:50℃2min,95℃10min;95℃30sec,60℃30sec,40cycles;绘制溶解曲线,最终数据以2-△△Ct进行分析。反应体系见表2。
表2实时荧光定量PCR反应体系
结果如图1所示,早期妊娠蜕膜组织中miR-29a表达水平最高,其次为早期流产蜕膜组织,再次为***分泌期子宫内膜组织,***增殖期子宫内膜组织中miR-29a表达水平最低。分泌期子宫内膜相比增生期子宫内膜miR-29a显著上调。与正常早期妊娠相比,早期流产蜕膜组织miR-29a的表达显著降低。
实施例2:人原代ESCs的分离培养及蜕膜化模型诱导
取人子宫组织,移至超净工作台内,放入含双抗PBS中;小心清理***多余组织,清洗血污,轻轻刮除表面黏膜,分离子宫内膜层组织,将组织剪成约1mm3碎块。PBS清洗组织一次,收集组织于离心管内,加入消化液于37℃水浴摇床消化,40min后加入完全培养基终止消化,用吸管反复吹打消化液,消化液经300g离心5min,丢弃上清,保留细胞沉淀。
(1)细胞培养:
用人ESCs完全培养基重悬细胞沉淀,接种于多聚赖氨酸预先包被的培养皿中,于37℃,5%CO2恒温培养箱中静置培养,48h后首次换液,以后每3天换液1次,细胞长满后待用。
(2)体外蜕膜化诱导:
待ESCs长到60%左右,进行体外蜕膜化。原代人子宫内膜基质细胞在含2%FBS,***E2(10nM)、甲孕酮P4(1μM)、cAMP(0.5mM)培养基培养5天诱导蜕膜化,miR-29a抑制剂在诱导前24h进行处理。蜕膜化形态学标志:成纤维的梭形子宫内膜***的形态变为大、圆且形状不规则的蜕膜细胞;蜕膜化分子标志物PRL、IGFBP-1表达。
实施例3:CCK-8检测miR-29a抑制剂对蜕膜化诱导ESCs细胞增殖的影响
(1)细胞培养:
将细胞置于含10%胎牛血清和1%双抗(100U/ml青链霉素)人ESCs完全培养基中,于37℃、含5%CO2、饱和湿度的培养箱中培养。2~3d更换培养基1次,5~6d用0.25%胰酶消化传代。
(2)实验分组:
①对照组;
②蜕膜化组;
③蜕膜化+抑制剂对照组;
④蜕膜化+miR-29a抑制剂组。
待ESCs长到60%左右,进行体外蜕膜化。原代人子宫内膜基质细胞在含2%FBS,***E2(10nM)、甲孕酮P4(1μM)、cAMP(0.5mM)培养基培养5天诱导蜕膜化,miR-29a抑制剂在诱导前24h进行处理。
(3)细胞增殖检测:
取处于对数生长期,生长状态良好的细胞,胰酶消化收集细胞,用培养基将细胞密度调整到5×104/mL,接入96孔板,每孔100μL细胞悬液,同时设空白组(在细胞孔周围孔内加入100μL无菌PBS),37℃、5%CO2培养箱中培养过夜;待细胞贴壁后,37℃培养箱中将细胞分组培养5天,每孔加入10μLCCK-8,37℃培养4h;酶标仪测定各孔吸光值OD 450。
如图3所示,与对照组相比,蜕膜化后子宫内膜基质细胞增殖活力增加;抑制剂对照组对蜕膜化诱导的子宫内膜基质细胞增殖活力基本无影响,而miR-29a抑制剂显著抑制蜕膜化诱导状态下子宫内膜基质细胞增殖活性(p<0.01)。结果表明,降低miR-29a水平显著抑制蜕膜化诱导的子宫内膜基质细胞增殖活力增加。
Claims (1)
1.一种用于检测miRNA标志物的试剂在制备早期流产诊断药物中的用途,其特征在于:所述标志物为hsa-miR-29a-3p。
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