miR-146b-5p downregulates IRAK1 and ADAM19 to suppress trophoblast proliferation, invasion and migration in miscarriage [circRNA, lincRNA, and mRNA profiles]

A large proportion of miscarriages are classified as unexplained miscarriages (UM) since no cause is identified. No reliable biomarkers or treatments are available for these pregnancy losses. While our transcriptomic sequencing has revealed substantial upregulation of miR-146b-5p in UM villous tissues, its role and associated molecular processes have yet to be fully characterized. Our work revealed that relative to samples from normal pregnancy (NP), miR-146b-5p was significantly elevated in villous tissues from UM patients and displayed promising diagnostic potential. Moreover, miR-146b-5p agomir contributed to higher rates of embryonic resorption in ICR mice. When overexpressed in HTR-8/SVneo cells, miR-146b-5p attenuated the proliferative, invasive, and migratory activity of these cells while suppressing the expression of MMP9 and immune inflammation-associated cytokines, including IL1B, IL11, CXCL1, CXCL8, and CXCL12. Conversely, inhibition of its expression enhanced proliferation, migration, and invasion abilities. Mechanistically, IRAK1 (IL-1 receptor-associated kinase-1) and ADAM19 (a disintegrin and metalloproteinase 19) were identified as miR-146b-5p targets regulating trophoblast function, and silencing IRAK1 had similar effects as miR-146b-5p overexpression, while IRAK1 overexpression could partially reverse the inhibitory impact of this miRNA on trophoblasts. miR-146b-5p may inhibit trophoblast proliferation, migration, invasion, and implantation-associated inflammation by downregulating IRAK1 and ADAM19, participating in the pathogenesis of miscarriage and providing a critical biomarker and a promising therapeutic target for UM. Maternal and gestational age-matched placental villus tissues from normal pregnancy (NP, n=5) and recurrent miscarriage (RM, n=5) were collected for RNA-seq of the whole transcriptome (including circRNA, lincRNA, miRNA and mRNA profiles) . To minimize the effect of maternal age and gestational age on miscarriage incidence or gene expression, specific criteria were used to match villus tissues from UM with those from NP. These criteria included a difference in maternal age ≤ 2 years and a difference in gestational age, as determined by ultrasound of ≤ 1 week.

由于未检出明确致病诱因，大部分流产被归类为不明原因流产（unexplained miscarriages, UM）。目前针对此类妊娠丢失，尚无可靠的生物标志物或治疗方案。我们的转录组测序研究显示，不明原因流产患者的绒毛组织中miR-146b-5p表达显著上调，但其具体功能及相关分子过程尚未完全阐明。本研究发现，相较于正常妊娠（normal pregnancy, NP）样本，不明原因流产患者绒毛组织中的miR-146b-5p水平显著升高，且具备良好的诊断应用潜力。此外，向ICR小鼠体内注射miR-146b-5p激动剂（agomir）后，小鼠胚胎吸收率显著升高。当在HTR-8/SVneo细胞中过表达miR-146b-5p时，该微小RNA可减弱细胞的增殖、侵袭及迁移能力，并抑制MMP9以及IL1B、IL11、CXCL1、CXCL8和CXCL12等免疫炎症相关细胞因子的表达。反之，抑制miR-146b-5p的表达则可增强细胞的增殖、迁移与侵袭能力。从机制层面分析，IRAK1（IL-1 receptor-associated kinase-1）与ADAM19（a disintegrin and metalloproteinase 19）被鉴定为miR-146b-5p调控滋养层细胞功能的靶基因；沉默IRAK1的生物学效应与miR-146b-5p过表达相似，而过表达IRAK1则可部分逆转该微小RNA对滋养层细胞的抑制作用。miR-146b-5p可能通过下调IRAK1与ADAM19的表达，抑制滋养层细胞的增殖、迁移、侵袭及着床相关炎症反应，参与流产的发病进程，并为不明原因流产提供了关键的生物标志物及潜在治疗靶点。本研究收集了年龄与孕周匹配的正常妊娠（normal pregnancy, NP，n=5）及复发性流产（recurrent miscarriage, RM，n=5）患者的胎盘绒毛组织，用于开展全转录组RNA测序，涵盖环状RNA（circRNA）、基因间长链非编码RNA（lincRNA）、miRNA及mRNA表达谱分析。为尽可能降低母亲年龄与孕周对流产发生率及基因表达的干扰，我们采用特定标准对不明原因流产患者与正常妊娠者的绒毛组织进行匹配：母亲年龄差值≤2岁，超声检测所得孕周差值≤1周。