miR-146b-5p downregulates IRAK1 and ADAM19 to suppress trophoblast proliferation, invasion, and migration in miscarriage†.

A large proportion of miscarriages are classified as unexplained miscarriages 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 unexplained miscarriage villous tissues, its role and associated molecular processes have yet to be fully characterized. Our work revealed that relative to samples from normal pregnancy, miR-146b-5p was significantly elevated in villous tissues from unexplained miscarriage 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, IL-1 receptor-associated kinase-1 and a disintegrin and metalloproteinase 19 were identified as miR-146b-5p targets regulating trophoblast function, and silencing IL-1 receptor-associated kinase-1 had similar effects as miR-146b-5p overexpression, while IL-1 receptor-associated kinase-1 overexpression could partially reverse the inhibitory impact of this microRNA on trophoblasts. miR-146b-5p may inhibit trophoblast proliferation, migration, invasion, and implantation-associated inflammation by downregulating IL-1 receptor-associated kinase-1 and a disintegrin and metalloproteinase 19, participating in the pathogenesis of miscarriage and providing a critical biomarker and a promising therapeutic target for unexplained miscarriage.

MiRNA-494 induces trophoblast senescence by targeting SIRT1.

OBJECTIVE: Although the mechanism underlying preeclampsia (PE) has been widely explored, the mechanisms related to senescence have not yet been fully revealed. Therefore, we investigated the role of the miR-494/longevity protein Sirtuin 1 (SIRT1) axis in PE. METHODS: Human placental tissue was obtained from severe preeclampsia (SPE) (n = 20) and gestational age-matched normotensive pregnancies (n = 20), and senescence-associated ß-galactosidase (SAßG) and SIRT1 expression levels were measured. The TargetScan and miRDB databases predicted candidate miRNAs targeting SIRT1, and intersected with differentially expressed miRNAs in the GSE15789 dataset (p < 0.05, |log2FC|≥1.5). Subsequently, we showed that miRNA (miR)-494 expression was significantly elevated in SPE, revealing miR-494 as a candidate SIRT1-binding miRNA. A dual-luciferase assay confirmed the targeting relationship between miR-494 and SIRT1. The senescence phenotype, migration, cell viability, reactive oxygen species (ROS) production levels and inflammatory molecule expression levels were measured after miR-494 expression was altered. We conducted a rescue experiment using SIRT1 plasmids to further demonstrate the regulatory relationship. RESULTS: SIRT1 expression was lower(p < 0.01) and miR-494 expression was higher (p < 0.001) in SPE, and SaßG staining showed premature placental aging in SPE (p < 0.001). Dual-luciferase reporter assays revealed that miR-494 targeted SIRT1. Compared to control cells, HTR-8/SVneo cells with upregulation of miR-494 had remarkably downregulated SIRT1 expression (p < 0.001), more SAßG-positive cells (p < 0.001), cell cycle arrested (p < 0.05), and upregulated P21 and P16 expression (p < 0.01). miR-494 overexpression also decreased HTR-8/SVneo cell migration (p < 0.05) and ATP synthesis (p < 0.001), increased ROS levels (p < 0.001), and upregulated NLRP3 and IL-1ß expression (p < 0.01). SIRT1-overexpressing plasmids partially reversed the effects of miR-494 overexpression in HTR-8/SVneo cells. CONCLUSION: The miR-494/SIRT1 interaction plays a role in the mechanism of premature placental aging in PE patients.

Upregulation of miRNA-26a Enhances the Apoptosis of Cerebral Neurons by Targeting EphA2 and Inhibiting the MAPK Pathway.

Neural tube defects (NTDs) constitute the second most common congenital malformation of the central nervous system. The pathogenesis of NTDs is not entirely clear. In recent years, microRNAs (miRNAs) have become a hot spot in genetic and developmental biology research. The present study aimed to explore the potential role of miRNA-26a in NTDs and the underlying pathogenesis thereof. First, we found significantly increased miRNA-26a expression in fetuses with NTDs (p < 0.0001), which significantly downregulated EphA2 and ERK1 mRNA and protein expression levels in fetuses with NTDs compared to normal controls (p < 0.01). In addition, a dual-luciferase reporter assay showed that miR-26a negatively regulated EphA2 by directly binding with the 3'-untranslated region of EphA2. Second, the upregulation of miRNA-26a expression increased caspase 3 and 9 protein expression levels (p < 0.01) and decreased EphA2 mRNA and protein expression levels (p < 0.01), as well as ERK1 and SRF protein expression levels (p < 0.01) in mouse neural stem cells (NE-4C) and human astroblastoma cells (U87MG). Furthermore, the upregulation of miRNA-26a inhibited cell proliferation and enhanced apoptosis of NE-4C and U87MG cells (p < 0.05). Similar results were observed with the MAPK inhibitor PD98059 (p < 0.01). These results suggest that miR-26a targets EphA2, modulates phosphorylation of the MAPK/ERK (MEK) pathway, regulates SRF, and participates in regulating nervous cell proliferation and apoptosis. Dysregulation of the aforementioned mechanism may be involved in the pathogenesis of NTDs.

MiR-135a-5p suppresses trophoblast proliferative, migratory, invasive, and angiogenic activity in the context of unexplained spontaneous abortion.

BACKGROUND: Spontaneous abortions (SA) is amongst the most common complications associated with pregnancy in humans, and the underlying causes cannot be identified in roughly half of SA cases. We found miR-135a-5p to be significantly upregulated in SA-associated villus tissues, yet the function it plays in this context has yet to be clarified. This study explored the function of miR-135a-5p and its potential as a biomarker for unexplained SA. METHOD: RT-qPCR was employed for appraising miR-135a-5p expression within villus tissues with its clinical diagnostic values being assessed using ROC curves. The effects of miR-135a-5p in HTR-8/SVneo cells were analyzed via wound healing, Transwell, flow cytometry, EdU, CCK-8, and tube formation assays. Moreover, protein expression was examined via Western blotting, and interactions between miR-135a-5p and PTPN1 were explored through RIP-PCR, bioinformatics analyses and luciferase reporter assays. RESULTS: Relative to normal pregnancy (NP), villus tissue samples from pregnancies that ended in unexplained sporadic miscarriage (USM) or unexplained recurrent SA (URSA) exhibited miR-135a-5p upregulation. When this miRNA was overexpressed in HTR-8/SVneo cells, their migration, proliferation, and cell cycle progression were suppressed, as were their tube forming and invasive activities. miR-135a-5p over-expression also downregulated the protein level of cyclins, PTPN1, MMP2 and MMP9. In RIP-PCR assays, the Ago2 protein exhibited significant miR-135a-5p and PTPN1 mRNA enrichment, and dual-luciferase reporter assays indicated PTPN1 to be a bona fide miR-135a-5p target gene within HTR-8/SVneo cells. CONCLUSION: miR-135a-5p may suppress trophoblast migratory, invasive, proliferative, and angiogenic activity via targeting PTPN1, and it may thus offer value as a biomarker for unexplained SA.

Analysis of circRNAs and circRNA-associated competing endogenous RNA networks in ß-thalassemia.

The involvement of circRNAs in ß-thalassemia and their actions on fetal hemoglobin (HbF) is unclear. Here, the circRNAs in ß-thalassemia carriers with high HbF levels were comprehensively analyzed and compared with those of healthy individuals. Differential expression of 2183 circRNAs was observed and their correlations with hematological parameters were investigated. Down-regulated hsa-circRNA-100466 had a strong negative correlation with HbF and HbA2. Bioinformatics was employed to construct a hsa-circRNA-100466­associated competing endogenous RNA (ceRNA) network to identify hub genes and associated miRNAs. The hsa-circRNA-100466▁miR-19b-3p▁SOX6 pathway was identified using both present and previously published data. The ceRNA network was verified by qRT-PCR analysis of ß-thalassemia samples, RNA immunoprecipitation of K562 cell lysates, and dual-luciferase reporter analysis. qRT-PCR confirmed that hsa-circRNA-100466 and SOX6 were significantly down-regulated, while miR-19b-3p was up-regulated. Hsa-circRNA-100466, miR-19b-3p, and SOX6 were co-immunoprecipitated by anti-argonaute antibodies, indicating involvement with HbF induction. A further dual-luciferase reporter assay verified that miR-19b-3p interacted directly with hsa-circRNA-100466 and SOX6. Furthermore, spearman correlation coefficients revealed their significant correlations with HbF. In conclusion, a novel hsa-circRNA-100466▁miR-19b-3p▁SOX6 pathway was identified, providing insight into HbF induction and suggesting targets ß-thalassemia treatment.

Placental transcriptome sequencing combined with bioinformatics predicts potential genes and circular RNAs associated with hemoglobin Bart's hydrops fetalis syndrome.

AIM: Hemoglobin Bart's hydrops fetalis syndrome (BHFS) is the most severe form of α-thalassemia. Histological alternations can be observed in placenta, but placental transcriptome profile and circular RNAs have not been studied in this disease. The aim of this study was to define the placental transcriptional changes and find relevant circular RNAs in BHFS. METHODS: We performed high-throughput RNA sequencing to detect placental samples from fetuses affected by BHFS (n = 5) and normal fetuses (NF, n = 5), quantitative reverse transcription polymerase chain reaction (RT-qPCR), and Sanger sequencing to validate the differentially expressed circRNAs and their potentially related miRNAs (BHFS, n = 22; NF, n = 11). Bioinformatics methods were performed for further analysis. RESULTS: Our results showed 152 differentially expressed genes (DEGs), 112 circRNAs, and 45 microRNAs that were differentially expressed. DEGs were found to be involved in Gene Ontology terms related to gas transport, cell adhesion, oxidative stress, organ development, hemopoiesis, and others. RT-qPCR results showed that hsa_circ_0003961 and hsa_circ_0006687 were upregulated (p < 0.05). The competing endogenous RNA and co-expression networks showed that hsa_circ_0003961 and hsa_circ_0006687 were connected with 3 miRNAs and some DEGs, including cell adhesion genes (e.g., CLDN19), hemoglobin related genes (e.g., SOX6 and HBZ) and angiogenesis related genes (e.g., EPHB2). Downregulations of hsa-miR-1299 and hsa-miR-625-5p in ceRNA network were also validated by RT-qPCR. Gene set enrichment analysis results for the two circRNAs showed that some gene sets associated with cell adhesion, hematopoietic system and apoptosis were significantly enriched. CONCLUSIONS: Our study characterized the placental transcriptome of BHFS. The circRNAs hsa_circ_0003961 and hsa_circ_0006687 in placenta may be relevant to BHFS.

Human m6A-mRNA and lncRNA epitranscriptomic microarray reveal function of RNA methylation in hemoglobin H-constant spring disease.

The thalassemia of Hemoglobin H-Constant Spring disease (HbH-CS) is the most common type of Thalassemia in non-transfusion thalassemia. Interestingly, the clinical manifestations of the same genotype of thalassemia can be vastly different, likely due to epigenetic regulation. Here, we used microarray technology to reveal the epigenetic regulation of m6A in modifiable diseases and demonstrated a role of BCL2A1 in disease regulation. In this study, we revealed that methylating enzyme writers including METTL16, WTAP, CBLL1, RBM15B, and ZC3H13 displayed low expression and the demethylating enzyme ALKBH5, along with reader proteins including IGF2BP2 and YTHDF3 exhibited high expression. In addition, BCL2A1 was hypo-methylated and showed low expression. We also revealed that the BCL2A1 methylation level and IGF2BP2 expression were negatively correlated. Additionally, the mRNAs expression between ALKBH5 and IGF2BP2 were positively correlated. In HbH-CS, most genes were hypo-methylated. This included BCL2A1, which may play an important role in the process of red blood cell differentiation and development of HbH-CS. Moreover, the mRNA-M6A methylation status may be regulated by the demethylating enzyme ALKBH5 via IGF2BP2.

Increased miRNA-518b inhibits trophoblast migration and angiogenesis by targeting EGR1 in early embryonic arrest†.

Evidence indicates that microRNAs (miRNAs) play essential roles in early embryonic development. The miRNA-518 family is a special biomarker of the placenta, and miRNA-518b is abnormally expressed in placental tissue in preeclampsia. Early growth response protein 1 (EGR1), a zinc finger transcriptional factor, plays an essential role in regulating cell differentiation, angiogenesis, and migration. Moreover, earlier studies have shown that EGR1 protein plays a key role in implantation. However, little is known about the role of miR-518b and EGR1 on early embryonic arrest (EEA) in humans. In our study, increased miR-518b along with decreased EGR1 was found in human villus tissues with EEA. Furthermore, we demonstrated by luciferase assay that miR-518b is a direct regulator of EGR1. After comparing the effect of silencing EGR1, vascular endothelial growth factor (VEGF) individually, and EGR1/VEGF in combination, we found that EGR1 can inhibit migration and angiogenesis of HTR-8 SVneo cells by decreasing the VEGF expression. Hypoxia plays an initial role in early embryonic development, and we found that hypoxia reduces the expression of miR-518b and increases the expression of EGR1 and VEGF to facilitate migration and angiogenesis in a hypoxic model of HTR-8/SVneo cell line. Our findings provide new insights into the role of miR-518b in EEA and implicate the potential application of miR-518b in the diagnosis and development of intervention for EEA.

High-throughput transcriptome-Seq and small RNA-Seq reveal novel functional genes and microRNAs for early embryonic arrest in humans.

Early Embryonic Arrest (EEA) is one of the major causes of female infertility. Genetic factors including specific genes and miRNAs may play pivotal roles on EEA. However, it is not well defined what genes and micro RNAs participate the pathophysiological alterations of EEA. In this work, we compared the Transcriptome -Seq and microRNA profiles from three pairs of villi (three EEA patients and three normal pregnancy, NP). We first confirmed the array data by qPCR with ten randomly selected differentially expressed genes and ten differentially expressed miRNAs in villi from 20 EEA and 20 NP controls. We next applied Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway analysis and found that these differentially expressed genes enriched in the PI3K-Akt signaling pathway, Jak-STAT signaling pathway, MAPK signaling pathway, Complement and coagulation cascades, Hypertrophic cardiomyopathy (HCM), Dilated cardiomyopathy (DCM). Interestingly, hsa-miR-6515-5p and its target genes NLRP3, UGP2 may regulate the Immune system and carbohydrate metabolism. Hsa-miRNA 518 and its target gene EGR1 may regulate cell proliferation, angiogenesis, and cell apoptosis to impact early embryonic development. Moreover, novel-m0045-5p and its target gene RMDN3 may regulate microtubule formation on the development of EEA. Our research provides novel biomarkers for EEA and establishes a foundation for further study of the mechanism of EEA.