The discovery of cell free fetal microRNAs (miRNAs) in maternal circulation has opened up novel probabilities for non-invasive prenatal diagnosis. This study aims to investigate circulating miRNAs as potential biomarkers in the serum of pregnant women with congenital heart defect (CHD) fetuses. A total of 110 pregnant women including 50 CHD cases and 60 healthy controls were included in this study. miRNA microarray followed by real-time PCR was used to explore miRNA expression. Receiver operating characteristic (ROC) curves were calculated to assess the diagnostic capability of miRNAs for fetal CHDs. 38 Serum miRNAs were revealed to be differentially expressed in the CHD group as compared to control group via microarray. Among these, nine down-regulated and three up-regulated miRNAs were validated by real-time PCR. Ten of these miRNAs were rapidly reduced in the normal maternal serum after delivery as compared to before delivery. In particular, we identified a biomarker panel consisting of four miRNAs (miR-142-5p, miR-1275, miR-4666a-3p and miR-3664-3p) capable of distinguishing CHDs from controls (area under the ROC curve (AUC), 0.920; p < 0.0001). The discovery of these dysregulated pregnancy-associated miRNAs in maternal serum may be potential biomarkers for non-invasive prenatal diagnosis of fetal CHDs.

Impaired autophagy and excessive apoptosis disrupt cellular homeostasis and contribute to neural tube defects (NTDs), which are a group of fatal and disabling birth defects caused by the failure of neural tube closure during early embryonic development. However, the regulatory mechanisms underlying NTDs and outcomes remain elusive. Here, we report the role of the transcription factor nuclear factor I-C (NFIC) in maintaining cellular homeostasis in NTDs. We demonstrated that abnormally elevated levels of NFIC in a mouse model of NTDs can interact with the miR-200b promoter, leading to the activation of the transcription of miR-200b, which plays a critical role in NTD formation, as reported in our previous study. Furthermore, miR-200b represses autophagy and triggers apoptosis by directly targeting the autophagy-related gene Ambra1 (Autophagy/Beclin1 regulator 1). Notably, miR-200b inhibitors mitigate the unexpected effects of NFIC on autophagy and apoptosis. Collectively, these results indicate that the NFIC-miR-200b-Ambra1 axis, which integrates transcription- and epigenome-regulated miRNAs and an autophagy regulator, disrupts cellular homeostasis during the closure of the neural tube, and may provide new insight into NTD pathogenesis.

Congenital heart defects (CHDs) are the most common group of major birth defects. Presently there are no clinically used biomarkers for prenatally detecting CHDs. Here, we performed a comprehensive maternal serum proteomics assessment, combined with immunoassays, for the discovery of non-invasive biomarkers for prenatal diagnosis of CHDs. A total of 370 women were included in this study. An isobaric tagging for relative and absolute quantification (iTRAQ) proteomic approach was used first to compare protein profiles in pooled serum collected from women who had CHD-possessing or normal fetuses, and 47 proteins displayed significant differential expressions. Targeted verifications were performed on 11 proteins using multiple reaction monitoring mass spectrometry (MRM-MS), and the resultant candidate biomarkers were then further validated using ELISA analysis. Finally, we identified a biomarker panel composed of 4 cytoskeletal proteins capable of differentiating CHD-pregnancies from normal ones [with an area under the receiver operating characteristic curve (AUC) of 0.938, P < 0.0001]. The discovery of cytoskeletal protein changes in maternal serum not only could help us in prenatal diagnosis of CHDs, but also may shed new light on CHD embryogenesis studies.

Neural tube defects (NTDs) are complex abnormalities associated with gene-environment interactions. The underlying cause has not been determined. Spinal cord tissues from cases with NTDs and healthy controls were collected. Methylation patterns between cases and normal individuals were compared using 450K Infinium Methylation BeadChip Illumina. DNA methylation analysis by pyrosequencing (PyroMark Q96) and mRNA and protein expression were analyzed using real-time quantitative PCR and Western blotting, respectively. Next-generation and Sanger sequencing were used to determine genetic variants in the target genes. Spinal cord tissues from cases with NTDs had more hypomethylated than hypermethylated genes. Further evaluation showed that the exon 1 region of TRIM4 was hypomethylated, and TRIM4 mRNA and protein levels were significantly increased in NTDs compared to controls. A rare missense variant (rs76665876) in TRIM4 was found in 3 of the 14 NTD cases but was not related to TRIM4 expression. TRIM4 mRNA levels were significantly increased in cases with hypomethylation and without the rs76665876 variant. These findings suggest that spinal cord tissues in cases with NTDs had a different genome-wide methylation pattern compared to controls. Abnormal methylation patterns in TRIM4 in immunity pathways might be involved in NTD pathogenesis. Genetic variants in TRIM4 genes only slightly contribute to the etiology of human NTDs.

Spinal bifida aperta (SBA) is a congenital malformation with a high incidence. Bone marrow mesenchymal stem cell (BMSC) transplantation has the potential to repair the structure of damaged tissues and restore their functions. This is an optional treatment that can be used as a supplement to surgery in the treatment of SBA. However, the application of BMSCs is limited, as the neuronal differentiation rate of BMSCs is not satisfactory when used in treating severe SBA. Thus, we aimed to assess the effect of neural stem cell (NSC)-derived exosomes on BMSC neuronal differentiation and observe the therapeutic effect in an ex vivo rat SBA embryo model. We found that NSC-derived exosomes increased the neuronal differentiation rate of BMSCs in vitro and in the SBA embryo model ex vivo. Proteomic analysis showed that NSC-derived exosomes were enriched in Netrin1, which positively regulated neuronal differentiation. Netrin1 increased the neuronal differentiation rate of BMSCs and NSCs and upregulated the expression of the neuronal markers, microtubule-associated protein (Map2), neurofilament, and β3-tubulin. Bioinformatic analysis revealed that Netrin1 treatment increased the expression of the transcription factors Hand2 and Phox2b, related to neuronal differentiation. Furthermore, the Netrin1-induced NSC neuronal differentiation was significantly blocked by Phox2b knockdown. We suggest that NSC-derived exosomal Netrin1 induces neuronal differentiation via the Hand2/Phox2b axis by upregulating the expression of Hand2 and Phox2b. Therefore, NSC-derived exosomes are a critical inducer of BMSC neuronal differentiation and represent a potential treatment agent that can benefit BMSC treatment in SBA.