microRNA-325-3p在体外受精-胚胎移植反复自然流产中的作用及机制

The effect and mechanism of miR-325-3p on RSA in IVF-ET

目的研究在体外受精-胚胎移植(IVF-ET)患者中,microRNA-325-3p(miR-325-3p)是否参与反复自然流产的发生和发展,以及在其中的作用机制。方法选取2015年1月至2019年6月在唐山市妇幼保健院就诊的89例IVF-ET反复自然流产患者和202例正常妊娠行人工流产患者作为研究对象。获取其妊娠4~12周的流产组织。通过原位杂交及实时定量逆转录聚合酶链反应(qRT-PCR)比较IVF-ET反复自然流产患者组织与正常人工流产患者组织中miR-325-3p表达差异;通过生物信息学网站预测miR-325-3p的可能靶基因,采用免疫印迹(Western blot)、qRT-PCR技术在细胞及组织水平对其进行验证,探究两者在IVF-ET反复自然流产患者中的作用机制。结果原位杂交显示,IVF-ET反复自然流产患者组织均表达miR-325-3p强信号,而正常人工流产患者组织仅存在miR-325-3p弱表达(P<0.01),qRT-PCR进一步证实该结果。Western blot、qRT-PCR检测miR-325-3p模拟物、抑制物转染的细胞,证实叉头框转录因子p3(Foxp3)为miR-325-3p的靶基因之一,且Foxp3蛋白及mRNA水平的表达量被miR-325-3p模拟物显著降调,被抑制物显著上调。与正常人工流产患者组织比较,IVF-ET反复自然流产患者组织Foxp3蛋白及mRNA表达均显著下降,差异具有统计学意义(P<0.05)。结论 MiR-325-3p的其中一个靶基因是Foxp3,通过miR-325-3p增高进而降调Foxp3的表达,来实现免疫耐受降低,可能是IVF-ET患者发生反复自然流产的机制之一。

Objective To investigate the effect and mechanism of microRNA-325-3 p(miR-325-3 p) on RSA in patients of in vitro fertilization and embryo transfer(IVF-ET). Methods 89 IVF-ET patients with recurrent spontaneous abortion and 202 normal pregnancy patients with induced abortion were selected from Tangshan Maternal and Child Health Care Hospital from January 2015 to June 2019. Aborted tissues from 4 to 12 weeks of gestation were obtained. The expression differences of miR-325-3 p in the tissues of IVF-ET patients with recurrent spontaneous abortion were compared by in situ hybridization and real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR). The possible target genes of miR-325-3 p were predicted on the bioinformatics website, and verified by Western blot and qRT-PCR at the cellular and tissue levels to explore the mechanism of miR-325-3 p in the patients with repeated spontaneous abortion in IVF-ET. Results In situ hybridization showed that miR-325-3 p was strongly expressed in the tissues of patients with repeated spontaneous abortion in IVF-ET, while was weakly expressed in the tissues of patients with normal induced abortion(P<0.01), which was further confirmed by qRT-PCR. Western blot and qRT-PCR were used to detect miR-325-3 p mimics and inhibitors in transfected cells, and confirmed that the forkhead box transcription factor P3(FoxP3) was one of the target genes of miR-325-3 p, and the expression levels of FoxP3 protein and mRNA were significantly down-regulated by miR-325-3 p mimics. The inhibition was significantly upregulated. Compared with the normal induced abortion patients, the expression of Foxp3 protein and mRNA in the tissues of IVF-ET patients with repeated spontaneous abortion were significantly decreased(P<0.05). Conclusions One of the target genes of miR-325-3 p is Foxp3, which can reduce the immune tolerance by increasing miR-325-3 p and then down-regulating the expression of Foxp3, which may be one of the mechanisms of repeated spontaneous abortion in IVF-ET patients.