miRNA-543 attenuates placental angiogenesis in female fetal growth restriction by targeting ANGPT2 and inhibiting the PI3K/AKT/mTOR pathway

Background Insufficient placental angiogenesis is a predominant pathological cause of fetal growth restriction (FGR). Placental capillary development is regulated in a sex-dependent manner, with female placentas exhibiting greater susceptibility to angiogenic dysfunction than male placentas. However, the sex-specific interplay between microRNAs (miRNAs) and human placental angiogenesis in FGR pathogenesis remains poorly defined. Emerging transcriptomic sequencing evidence has implicated miR-543 as a pivotal regulator of impaired angiogenesis in female FGR placentas. This study aimed to elucidate the sex-dependent mechanistic role of miR-543 in modulating placental angiogenesis in a cell-specific manner. Method FGR pregnancies were recruited, and placental tissues were collected after delivery. The dual-luciferase reporter assay was used to detect the direct binding site of miR-543 to ANGPT2. Next, immunohistochemistry (IHC) and immunofluorescence (IF) staining were used to detect the expression and localization of ANGPT2. To verify the cell-specific gene expression of miR-543 and ANGPT2, laser capture microdissection (LCM) was employed to isolate endothelial cells from human placentas. The gene expression levels of miR-543 and ANGPT2 were quantified by qRT-PCR. CCK8, wound healing, Transwell, and tube formation assays and flow cytometry were performed to assess the functional effects of miR-543 and ANGPT2 on the biological function of human umbilical vein endothelial cells (HUVECs). The PI3K/AKT/mTOR signaling pathway was predicted and verified by Western blotting. Results Significantly downregulated miR-543 expression was observed in the placentas of female FGR fetuses relative to matched normal controls. According to target gene prediction and the dual-luciferase reporter assay, ANGTP2 was identified as a target candidate of miR-543. Moreover, immunofluorescence staining and LCM analysis verified the cell-specific distribution of ANGPT2 in female FGR placentas, partly indicating its key involvement in placental capillary angiogenesis. Furthermore, dysregulated expression of miR-543 and ANGPT2 was closely correlated with aberrant proliferation, migration, and apoptosis of HUVECs. KEGG analysis revealed that the candidate targets of miR-543 were mainly enriched in the PI3K/AKT/mTOR pathway. Western blot analysis demonstrated that the PI3K/AKT/mTOR signaling pathway mediated the ANGPT2-induced attenuation of placental angiogenesis in female FGR. Conclusion miR-543 negatively regulated ANGPT2 expression, leading to impaired endothelial cell function via inactivation of the PI3K/AKT/mTOR signaling pathway. This, in turn, contributed to sex-specific inhibition of capillary angiogenesis in the placenta of female fetuses. These findings reveal a novel miRNA-mediated, sex-dependent regulatory mechanism underlying the pathogenesis of impaired placental angiogenesis.