NHC Key Lab of Reproduction Regulation (Shanghai Institute for Biomedical and Pharmaceutical Technologies), School of Pharmacy, Fudan University, Shanghai, 200032, China.
The Second Hospital of Tianjin Medical University, Tianjin, China.
Placenta. 2022 Aug;126:1-11. doi: 10.1016/j.placenta.2022.05.016. Epub 2022 May 28.
Increasing evidence has shown that circular RNAs (circRNAs) play vital roles in embryonic development. However, the function of circRNAs in recurrent spontaneous abortion (RSA) is largely unknown. This study aimed to investigate the expression profile of human circRNAs and their functional mechanisms in regulating RSA.
The profiles of circRNAs in placental villus tissues from women with RSA and healthy pregnancy with induced abortion were investigated using RNA-sequencing and bioinformatics. Nine circRNAs were verified in the 50 placental villus samples. RNase R digestion, actinomycin D treatment, and fluorescence in situ hybridization were performed to characterize circFBXW4. Furthermore, direct binding of circFBXW4 to miR-324-3p was confirmed by dual-luciferase reporter assay. The roles of circFBXW4 were determined by loss- and gain-of-function assays including cell proliferation, invasion, and apoptosis using CCK8 kit, transwell migration assay, and TUNEL kit in vitro, respectively.
A total of 417 aberrantly expressed circRNAs was detected. circFBXW4, a circRNA significantly up-regulated in the RSA group, was further evaluated. circFBXW4 showed higher stability than FBXW4 mRNA and was localized in the cytoplasm and nucleus in HTR-8/SVneo cells. MiR-324-3p was lowly expressed in the RSA group, and directly regulated circFBXW4 and TJP1 expression in a targeted manner. Overexpression and knockdown of circFBXW4 and miR-324-3p mimic/inhibitor could increase or decrease HTR-8/SVneo cell proliferation and invasion. circFBXW4 regulated TJP1 expression, cell proliferation, and invasion by sponging miR-324-3p.
The circFBXW4/miR-324-3p/TJP1 axis is involved in the occurrence and progression of RSA and may be a promising therapeutic target in RSA.
越来越多的证据表明，环状 RNA（circRNA）在胚胎发育中发挥着重要作用。然而，circRNA 在复发性自然流产（RSA）中的功能很大程度上是未知的。本研究旨在探讨人类 circRNA 的表达谱及其在调节 RSA 中的功能机制。
采用 RNA-seq 和生物信息学方法检测 RSA 患者和人工流产健康妊娠妇女胎盘绒毛组织中 circRNA 的表达谱。在 50 个胎盘绒毛样本中验证了 9 个 circRNA。通过 RNase R 消化、放线菌素 D 处理和荧光原位杂交技术对 circFBXW4 进行了特征分析。此外，通过双荧光素酶报告基因实验证实了 circFBXW4 与 miR-324-3p 的直接结合。通过 CCK8 试剂盒、Transwell 迁移实验和 TUNEL 试剂盒分别进行细胞增殖、侵袭和凋亡的体外失活和激活功能实验，确定了 circFBXW4 的作用。
共检测到 417 个异常表达的 circRNA。circFBXW4 在 RSA 组中显著上调，进一步进行了评估。circFBXW4 比 FBXW4 mRNA 更稳定，并且在 HTR-8/SVneo 细胞中定位于细胞质和细胞核。miR-324-3p 在 RSA 组中表达较低，并且以靶向方式直接调节 circFBXW4 和 TJP1 的表达。过表达和敲低 circFBXW4 和 miR-324-3p 模拟物/抑制剂可增加或减少 HTR-8/SVneo 细胞的增殖和侵袭。circFBXW4 通过海绵 miR-324-3p 调节 TJP1 表达、细胞增殖和侵袭。
circFBXW4/miR-324-3p/TJP1 轴参与 RSA 的发生和发展，可能是 RSA 的有前途的治疗靶点。
