A new Prdm1-Cre line is suitable for studying the second heart field development.

The second heart field (SHF) plays a pivotal role in heart development, particularly in outflow tract (OFT) morphogenesis and septation, as well as in the expansion of the right ventricle (RV). Two mouse Cre lines, the Mef2c-AHF-Cre (Mef2c-Cre) and Isl1-Cre, have been widely used to study the SHF development. However, Cre activity is triggered not only in the SHF but also in the RV in the Mef2c-Cre mice, and in the Isl1-Cre mice, Cre activation is not SHF-specific. Therefore, a more suitable SHF-Cre line is desirable for better understanding SHF development. Here, we generated and characterized the Prdm1-Cre knock-in mice. In comparison with Mef2c-Cre mice, the Cre activity is similar in the pharyngeal and splanchnic mesoderm, and in the OFT of the Prdm1-Cre mice. Nonetheless, it was noticed that Cre expression is largely reduced in the RV of Prdm1-Cre mice compared to the Mef2c-Cre mice. Furthermore, we deleted Hand2, Nkx2-5, Pdk1 and Tbx20 using both Mef2c-Cre and Prdm1-Cre mice to study OFT morphogenesis and septation, making a comparison between these two Cre lines. New insights were obtained in understanding SHF development including differentiation into cardiomyocytes in the OFT using Prdm1-Cre mice. In conclusion, we found that Prdm1-Cre mouse line is a more appropriate tool to monitor SHF development, while the Mef2c-Cre mice are excellent in studying the role and function of the SHF in OFT morphogenesis and septation.

Comorbidities in congenital heart disease: different patterns in childhood and adulthood.

BACKGROUND: Existing studies were no exploration of the association between congenital heart disease (CHD) in children and comorbidities. This study was to assess the prevalence and number of comorbidities in CHD among children and adults, and to compare the comorbidity patterns by children and adults using association rule analysis. METHODS: Patients identified by the International Classification of Diseases, Ninth Revision (ICD-9) code in the Medical Information Mart for Intensive Care III (MIMIC-III) 2001-2012 and MIMIC-IV 2008-2018 were included in this cross-sectional study. Association rule analysis was used to explore associations between CHD and comorbidities in children and adults using values of support (%), confidence (%), and lift. RESULTS: Among 60,400 eligible patients, 1.54% of adults had CHD and 0.83% of adults had CHD with at least one comorbidity, 13.79% had CHD and 12.37% had CHD with at least one comorbidity in children. The most common comorbidities were circulatory system diseases (53.78%), endocrine diseases (35.76%), and respiratory system diseases (23.46%) in adults with CHD, and the most common comorbidities were perinatal diseases (87.50%) in children with CHD. The comorbidity rate was 90.19% and 56.68% in children and adults, respectively. In children, perinatal diseases, circulatory system diseases, and endocrine diseases had the highest prevalence. The incidence of circulatory system diseases, perinatal diseases and endocrine diseases in CHD adults was confidence = 31.56%, 36.11%, and 23.23%, respectively. Perinatal diseases were common comorbidities among all CHD severity groups in children and adults. CONCLUSION: The prevalence of comorbidities in children with CHD was higher than that in adults with CHD. The most common comorbidities were perinatal diseases and endocrine diseases among children and adults with CHD, respectively. Our study provided insights into comorbidity patterns in children and adults with CHD.

Incidence and Risk Factors for Retinopathy of Prematurity in a Tertiary Hospital in China.

Purpose: To investigate the incidence and risk factors for the retinopathy of prematurity (ROP) in the neonatal intensive care unit (NICU) of Obstetrics and Gynecology Hospital of Affiliated to Nanjing Medical University, China. Methods: This retrospective case-control study included 611 preterm infants with birth weight (BW)<1500 grams admitted to the Department of Neonatology, Obstetrics and Gynecology Hospital of Affiliated to Nanjing Medical University between January 2019 and December 2022. The incidence and risk factors for any stage and severe ROP were analyzed. Results: Within 611 infants, 245(40.1%) developed ROP; 160(26.2%) infants were stage 1, 54(8.8%) were stage 2, and 31(5.1%) were stage 3, no stage 4 and 5. Among them, 22(3.6%) infants needed treatment. Multivariate analysis showed a higher gestational age (GA) was protective, whereas twin birth and moderate-to-severe BPD increased the hazard of any stage ROP; higher BW and male gender were significant risk factors for severe ROP. Conclusion: Compared to other tertiary hospitals, the incidence of any stage ROP in our NICU was higher, but the rate of ROP needed treatment was lower. A higher GA was protective, whereas twin birth and moderate-to-severe BPD increased the hazard of any stage ROP; higher BW was protective, whereas male gender were risk factors for the development of severe ROP.

Effect of First Mother's Own Milk Feeding Time on the Risk of Moderate and Severe Bronchopulmonary Dysplasia in Infants With Very Low Birth Weight.

Objective: To explore the effect of mother's own milk (MOM) feeding time on the risk of moderate and severe bronchopulmonary dysplasia (BPD) in infants with very low birth weight (VLBW). Methods: Clinical data from 630 infants with VLBW were retrospectively analyzed. Participants were divided into the early mother's own milk (EMOM) feeding group (first mother's own milk feeding time ≤72 h after birth, n = 397) and the late mother's own milk (LMOM) feeding group (first mother's own milk feeding time >72 h after birth, n = 233). Differences in the incidence of moderate and severe BPD among the two groups were analyzed using the chi-square test. Effects of MOM feeding time on the incidence of moderate and severe BPD were evaluated using univariate and multivariate logistic regression analysis. Results: The incidences of moderate and severe BPD in the EMOM feeding group and the LMOM feeding group were 13.9% (55/397) and 21.0% (49/233), respectively (P = 0.019). Variate logistic regression analysis showed that the LMOM feeding group had an increased risk of moderate and severe BPD compared with the EMOM feeding group (OR = 1.656, 95% CI:1.083-2.532). The results of multivariate logistic regression analysis showed that the LMOM feeding group had an increased risk of moderate and severe BPD compared with the EMOM feeding group (OR = 1.894, 95% CI:1.127-3.185). Conclusion: The first time of MOM feeding within 72 h after birth and the persistence of mother's own milk feeding during hospitalization can reduce the incidence of moderate and severe BPD in infants with VLBW.

The role of polypeptide PDTLN1 in suppression of PI3K/AKT signaling causes cardiogenetic disorders in vitro and in vivo.

AIMS: A new polypeptide, PDTLN1, derived from the human Talin-1 protein, which is highly expressed in both myocardial tissue and maternal peripheral blood of aborted fetuses with congenital heart disease (CHD). However, its role in cardiac developmental disorders has not been disclosed till now. In the present study, we aim to assess the functions of PDTLN1 in heart development of zebrafish and cellular viability, proliferation, and apoptosis of P19 cells. MAIN METHODS: Cellular viability was assessed by Cell Counting Kit-8, the EdU Kit was used to evaluate cellular proliferation, and apoptosic rate of P19 was examined using FITC Annexin-V staining followed by flow cytometry. The zebrafish embryos were divided into three groups: PEP group and NC group were microinjected with polypeptides, WT group without any intervention. The protein expression of PI3K/AKT were evaluated by western blotting. KEY FINDINGS: PDTLN1 could suppress the proliferation, and facilitate apoptosis. PDTLN1 caused abnormal heart development of zebrafish embryos and the PDTLN1 (50 µM)-injected group showed an aberrant expression pattern of vmhc, amhc and cmlc2. Compared to the CTL group and SC79 group of P19 cells, the PDTLN1 group had a lower phosphorylated PI3K/AKT proteins level, decreased cellular viability and lower proliferation activity. SIGNIFICANCE: PDTLN1 caused cardiac developmental defects in zebrafish, inhibited cellular viability, proliferation, and promoted apoptosis of P19 cells via suppressing the PI3K/AKT signaling pathway. Our findings provide a fresh perspective on the functional mechanism of human-derived peptides and may promote novel diagnostic biomarkers detection and therapeutic targets in CHD.

Using a WeChat mini-program-based lactation consultant intervention to increase the consumption of mother's own milk by preterm infants in the neonatal intensive care unit: a study protocol for a cluster randomized controlled trial.

BACKGROUND: The benefits of mother's own milk (MOM) for preterm infants have been widely recognized. Many studies have shown that the rate of breastfeeding of premature infants remains very low. Although many studies use measures to promote breastfeeding, few high-quality cluster randomized controlled studies have evaluated the effectiveness of these measures. WeChat is an instant messaging software for smart terminals, and WeChat mini-programs have been widely used to promote health and self-management in China. Based on this background, we designed a randomized controlled study based on WeChat mini-programs to promote MOM feeding of premature infants in the neonatal intensive care unit (NICU). METHODS/DESIGN: This study will evaluate the effectiveness of WeChat mini-programs to increase the consumption of MOM feeding in twelve NICUs in Jiangsu Province, namely, six "intervention" NICUs and six "control" NICUs. The study process is as follows: (1) design and preparation, (2) NICU recruitment and training, (3) interpretation and analysis of baseline data, (4) quality control implementation process, and (5) data analysis feedback and publication of study reports. The primary outcome is the proportion of MOM feeding of premature infants during NICU hospitalization. The secondary outcomes are as follows: (1) time to initiation of MOM feeding (hours) and proportion of first-time MOM feeding (%), (2) duration of parenteral nutrition (days), (3) time to total gastrointestinal feeding (days), (4) hospitalization time and hospitalization cost, and (5) incidence of complications (necrotizing enterocolitis, bronchopulmonary dysplasia, feeding intolerance, late-onset sepsis, retinopathy of prematurity). DISCUSSION: This study is the first cluster randomized controlled trial on the intervention of using a WeChat mini-program-based lactation consultant for premature infants in the NICU in China. We hope this study can improve the consumption of MOM by NICU premature infants during hospitalization through the intervention of WeChat mini-programs. TRIAL REGISTRATION: ClinicalTrials.gov NCT04383379. Registered on May 5, 2020.

Circular RNA hsa_circ_105039 promotes cardiomyocyte differentiation by sponging miR­17 to regulate cyclinD2 expression.

Previously it was found that hsa_circ_105039 was underexpressed in the heart tissue of patients with congenital heart disease (CHD). However, the function and mechanism of hsa_circ_105039 in CHD are unclear. In the present study, induced pluripotent stem (iPS) cells were differentiated into cardiomyocytes using 1% dimethyl sulfoxide (DMSO). Cell differentiation, viability, migration and apoptosis were measured before and following hsa_circ_105039 knockdown or overexpression. The results indicated that hsa_circ_105039 overexpression promoted cell differentiation, viability and migration; whereas apoptosis was simultaneously repressed. A luciferase reporter assay verified that hsa_circ_105039 acted as a sponge for microRNA (miR)­17 and that cyclinD2 was a direct target of miR­17. Furthermore, differentiation­related genes and proteins were analyzed by reverse transcription­quantitative PCR and western blotting, respectively. The results showed that hsa_circ_105039 could also upregulate the expression of differentiation­related genes and proteins, including natriuretic peptide A, cardiac troponin I, GATA­binding protein 4 and homobox transcription factor, in iPS cells. The results suggested that hsa_circ_105039 exerted a protective effect by promoting miR­17/cyclinD2 in DMSO­induced iPS cardiomyocytes, which indicated that hsa_circ_105039 is a potential key molecule for the diagnosis of CHD.

[A clinical analysis of sepsis in very low birth weight infants].

OBJECTIVE: To study the incidence and clinical features of sepsis in very low birth weight (VLBW) infants. METHODS: The clinical data were collected from VLBW infants, with a birth weight of < 1 500 g, who were admitted to the Department of Neonatology, Maternity Hospital Affiliated to Nanjing Medical University, from January 2019 to June 2020. The incidence of sepsis, distribution of pathogenic bacteria, and risk factors for sepsis were analyzed. RESULTS: A total of 369 infants were enrolled, and 138 infants had sepsis, among whom 84 had early-onset sepsis (EOS) and 54 had late-onset sepsis (LOS). Enterococcus faecalis (24%) and Streptococcus (21%) were the main pathogenic bacteria in infants with EOS, and Staphylococcus (41%) and Enterobacter (29%) were the main pathogenic bacteria in infants with LOS. The incidence of EOS and LOS decreased with the increase of gestational age and birth weight (P < 0.05). The multivariate logistic regression analysis showed that a high birth weight was a protective factor against EOS (OR=0.996, 95%CI:0.993-0.998, P < 0.05), while vaginal delivery (OR=2.781, 95%CI:1.190-6.500, P < 0.05) was a risk factor for EOS, and long duration of parenteral nutrition was a risk factor for LOS (OR=1.129, 95%CI:1.067-1.194, P < 0.05). CONCLUSIONS: Enterococcus faecalis is the most common pathogenic bacteria for EOS, and Staphylococcus is the most common pathogenic bacterium for LOS in VLBW infants. A high birth weight may reduce the risk of EOS in VLBW infants, while vaginal delivery may increase the risk of EOS. Prolonged parenteral nutrition may increase the risk of LOS.

Lipidomic Profiling of Human Milk Derived Exosomes and Their Emerging Roles in the Prevention of Necrotizing Enterocolitis.

SCOPE: Human milk can prevent the development of necrotizing enterocolitis (NEC). Human milk is rich in cargo-carrying exosomes that participate in intercellular communication. This study investigated the effects of term and preterm human milk-derived exosomes, and elucidated their lipid expression profiles. METHODS AND RESULTS: Milk from healthy mothers is collected who have delivered full-term or preterm infants, and exosomes are isolated and quantified. Administration of term and preterm milk exosomes significantly enhances epithelial proliferation and migration in vitro, and ameliorates the severity of NEC in vivo. A total of 395 lipids are identified in term and preterm human milk-derived exosomes. Bioinformatics analysis and western blotting reveal that top 50 lipids regulate intestinal epithelial cell function via the Extracellular-Signal-Regulated Kinase/Mitogen Activated Protein Kinase (ERK/MAPK) pathway. CONCLUSION: This study reveals for the first time the lipidomic complexities in exosomes derived from preterm and term milk. The results provide novel mechanistic insight on how human milk prevents the development of NEC.

[Influence of twin pregnancy by assisted reproductive technology on neonatal outcomes].

OBJECTIVE: To study the influence of twin pregnancy by assisted reproductive technology (ART) versus twin pregnancy by spontaneous conception (SC) on neonatal outcomes. METHODS: A retrospective analysis was performed for the clinical data of 3 356 live twins with a gestational age of ≥24 weeks who were born in Nanjing Maternal and Child Health Hospital from 2017 to 2019, with 2 006 twins (1 003 pairs) in the ART group and 1 350 (675 pairs) in the SC group. The two groups were compared in terms of the mother's general information and pregnancy comorbidities and the general information, diseases, and outcomes of neonates. RESULTS: Compared with the SC group, the ART group had a significantly higher maternal age (P < 0.05) and significantly higher rates of primiparity, cesarean section, and cervical cerclage (P < 0.05). Compared with the SC group, the ART group had significantly higher incidence rates of maternal pregnancy comorbidities including hypertension, gestational diabetes, and postpartum hemorrhage (P < 0.05). Compared with the SC group, the ART group had a significantly lower mean gestational age of neonates (P < 0.05) and a significantly higher proportion of very-low-birth-weight infants (6.8% vs 5.8%, P < 0.05), while ART did not increase the risks of preterm birth and low Apgar score. There were no significant differences between the two groups in the mortality rate of neonates and the incidence rates of neonatal diseases including respiratory distress syndrome, stage II/III necrotizing enterocolitis, bronchopulmonary dysplasia, and grade III-IV intracranial hemorrhage (P > 0.05). CONCLUSIONS: Compared with twin pregnancy by SC, twin pregnancy by ART does not increase the neonatal mortality rate and risk of adverse outcomes.

Transcriptomic analysis reveals the role of a peptide derived from CRYAB on the CoCl2-induced hypoxic HL-1 cardiomyocytes.

Acute myocardial infarction (AMI) is a life-threatening disease that often results in heart failure. CRYAB, a small heat shock protein, has been shown to have cardioprotective effects against oxidative stress-induced apoptosis in AMI. Previously, we purified a peptide derived from CRYAB (LEDQFFGEH), which we named PDFC. In this study, we determined the function of PDFC on HL-1 cardiomyocytes and explored the mechanism underlying its function. A hypoxic myocardiocyte cell line was generated by stimulation of HL-1 mouse cardiac muscle cells with different concentrations of CoCl2. Then, the hypoxic HL-1 cells were treated with the synthetic PDFC peptide, and cell proliferation, migration, and apoptosis were assessed to examine the effects of PDFC on HL-1 and hypoxic HL-1 cells. To examine the mechanism underlying the effects of PDFC on hypoxic cells, PDFC-treated hypoxic HL-1 cells were submitted for deep RNA sequencing. Finally, several differentially expressed genes in different pathways were selected for confirmation by RT-qPCR. Hypoxic myocardiocytes were generated by stimulating HL-1 cells with 800 µM CoCl2 for 24 h, which significantly upregulated HIF-1α. PDFC at 200 µg/ml showed the most positive effects on cell viability. Although hypoxic HL-1 cells and PDFC-treated hypoxic HL-1 cells both showed lower viability and migration and higher levels of apoptosis than untreated HL-1 cells, compared to hypoxic HL-1 cells, PDFC-treated hypoxic HL-1 cells showed higher viability and migration and lower apoptosis. The deep sequencing showed that 812 genes were upregulated and 1946 genes were downregulated. Among these differentially expressed genes, 699 of the upregulated genes and 1488 of the downregulated genes were protein-coding genes. Gene ontology and pathway enrichment analysis showed that the downregulated genes were dominant and that the PI3K-Akt pathway was located in the center of the network. A protein-protein interaction network was constructed, and 892 nodes were determined. In PDFC-treated hypoxic HL-1 cells, Fn1, Pik3r5, and Creb5 were downregulated, while Insr, Bcl2, Mapk14, and Pten were upregulated when compared to the levels in hypoxic HL-1 cells. In conclusion, this study reveals the significant bioactive effect of the CRYAB-derived peptide, PDFC on cardiomyocytes and the underlying mechanism.

MicroRNA-375 overexpression disrupts cardiac development of Zebrafish (Danio rerio) by targeting notch2.

MicroRNAs are small noncoding RNAs that are important for proper cardiac development. In our previous study of fetuses with ventricular septal defects, we discovered that microRNA-375 (miR-375) is obviously upregulated compared with that in healthy controls. Our study also confirmed that miR-375 is crucial for cardiomyocyte differentiation. This research mainly focused on the biological significance and mechanism of miR-375 using a zebrafish model. We injected zebrafish embryos with 1-2 nl of a miR-375 mimic at various concentrations (0/2/4/8 µM) or with negative control. The deformation and mortality rates were separately assessed. The different expression levels of miR-375 and related genes were examined by qRT-PCR, and luciferase assays and in situ hybridization were used to clarify the mechanism of miR-375 during embryonic development. Overexpression of miR-375 disrupted the cardiac development of zebrafish embryos. Disruption of miR-375 led to a decreased heart rate, pericardial edema, and abnormal cardiac looping. Various genes involved in cardiac development were downregulated due to the overexpression of miR-375. Moreover, the NOTCH signaling pathway was affected, and the luciferase reporter gene assays confirmed notch2, which was predicted by bioinformatics analysis, as the target gene of miR-375. Our findings demonstrated that the overexpression of miR-375 is detrimental to embryonic development, including cardiac development, and can partially simulate a multisystemic disorder. MiR-375 has an important role during cardiac morphogenesis of zebrafish embryos by targeting notch2, indicating its potential as a diagnostic marker.

Differential contribution of the two waves of cardiac progenitors and their derivatives to aorta and pulmonary artery.

During mouse development, part of the cells derived from the second heart field (SHF) progenitors contributes to the elongation and enlargement of the outflow tract (OFT) that subsequently septates into the trunks of aorta (Ao) and pulmonary artery (PA). Thus, the cardiac progenitor-originated cells are distributed to both Ao and PA. Here, we investigated that how these cells are assigned to the two great arteries during OFT septation through lineage tracing technology. By use of the inducible Mef2c-AHF-CreERT2; Rosa26-mTmG reporter system, two waves of SHF progenitors and their derivatives were identified, and they made differential contribution to the Ao and PA, respectively. While the early wave of cells (at E7.5) was preferentially destined to the Ao, the second wave of cells (from E8.5 till E11.5) made its favorite path to the PA. In addition, we unveiled PDK1 as a critical regulator of the second wave of cells as deletion of Pdk1 resulted in poorly developed PA leading to pulmonary stenosis. Thus, this study provides insights into the understanding of the pre-determined cell fate of the cardiac progenitor-derived cells with preferential contribution to the Ao and PA, as well as of the pathogenesis of pulmonary stenosis.

Profiles analysis reveals circular RNAs involving zebrafish physiological development.

Recent studies have found that known functions of circular RNAs (circRNAs) include sequestration of microRNAs (miRNAs) or proteins, modulation of transcription and interference with splicing, and even translation to produce polypeptides. The zebrafish model is also demonstrably similar to humans in many studies. To explore the changes in circRNAs during embryonic development and to further research the mechanism of action of circRNAs in development-related diseases, Zebrafish embryos at the blastula period, gastrula period, segmentation period, throat stage, and incubation period were collected. Illumina deep-sequencing technology and CircRNA Identifier (CIRI) algorithm were used to detect circRNAs. In total, we identified 1,028 circRNAs (junction reads ≥5 and p < 0.05). Considering that the function of circRNAs is related to host genes, a bioinformatics analysis revealed these differentially expressed host genes are involved in NOTCH signaling pathways, cardiovascular system development, retinal ganglion cell axon guidance, and so on. Moreover, circRNAs can participate in biological regulation through the function of miRNA sponges. TargetScan and miRanda were used to predict 73 miRNAs binding to circRNAs such as miR-19b, miR-124, and so on. Some miRNAs play important roles in embryogenesis. The peak expression of circRNAs is distributed at different time points, suggesting that it may be involved in embryogenesis at different stages. Our study provides a foundation for understanding the dynamic regulation of circRNA transcriptomes during embryogenesis and identifies novel key circRNAs that might control embryonic development in a zebrafish model.

PID1 in adipocytes modulates whole-body glucose homeostasis.

The novel obesity-associated protein Phosphotyrosine Interaction Domain containing 1 (PID1) inhibits insulin-PI3K/Akt signaling pathway and insulin-stimulated glucose uptake in vitro. In this study, we generated fat tissue-specific aP2-PID1 transgenic (aP2-PID1tg) mice and PID1 knockout (PID1-/-) mice to explore how PID1 affects glucose metabolism in vivo. We observed insulin resistance and impaired insulin-PI3K/Akt signaling in aP2-PID1tg mice. Consistent with these data, the PID1-/- mice displayed improved glucose tolerance and insulin sensitivity under chow diet, with increased Akt phosphorylation in white adipose tissue (WAT). We further demonstrated that PID1 could interact with low density lipoprotein receptor-related protein 1 (LRP1) but not the insulin receptor (IR) in adipocytes, and its overexpression could lead to decreased GLUT4 level. Our results thus indentify PID1 as a critical regulator of glucose metabolism in adipocytes.

Peptidomic Analysis of Maternal Serum to Identify Biomarker Candidates for Prenatal Diagnosis of Tetralogy of Fallot.

Tetralogy of Fallot (TOF) is the most common form of cyanotic congenital heart disease. To identify endogenous peptides possibly involved in the progression of TOF, we performed comparative peptidomic profiling of maternal serum between normal fetuses and fetuses suffering from TOF. A total of 278 differentially expressed peptides, including 94 over-expressed peptides and 184 under-expressed peptides, originating from 227 protein precursors were identified by liquid chromatography/mass spectrometry (LC/MS) in maternal serum of fetuses with TOF compared to normal controls. Further, ingenuity pathway analysis (IPA) was used to identify putative roles for these peptides in cardiovascular development. Two peptides were derived from functional domains of proteins involved in heart development and associated with TOF; these may represent candidate bioactive peptides involved in TOF. These peptides may be related to the pathologic changes in the heart associated with TOF, and may be useful as novel biomarkers for prenatal diagnosis of TOF. J. Cell. Biochem. 119: 468-477, 2018. © 2017 Wiley Periodicals, Inc.

Circulating LncRNAs as Novel, Non-Invasive Biomarkers for Prenatal Detection of Fetal Congenital Heart Defects.

OBJECTIVES: To explore the clinical value of circulating long non-coding RNAs (lncRNAs) as biomarkers to predict fetal congenital heart defects (CHD) in pregnant women. METHODS: Differential expression of lncRNAs isolated from the plasma of pregnant women with typical fetal CHD or healthy controls was analyzed by microarray. Gene ontology (GO), pathway and network analysis were performed to study the function of the lncRNAs. Differentially expressed lncRNAs were validated in plasma samples from 62 pregnant women with typical CHD and 62 matched controls by RT-PCR. The sensitivity and specificity of each lncRNA in the diagnosis of fetal CHD was determined by ROC curve analysis. RESULTS: Microarray analysis identified 3694 up-regulated and 3919 down-regulated (fold change ≥2.0) lncRNAs. The top ten significantly differentially expressed, CHD-associated lncRNAs were validated by RT-PCR. Five significantly up-regulated or down-regulated lncRNAs were identified: ENST00000436681, ENST00000422826, AA584040, AA709223 and BX478947 with the AUC of ROC curves calculated as 0.892, 0.817, 0.755, 0.882 and 0.886, respectively. CONCLUSIONS: Specific lncRNAs aberrantly expressed in the plasma of pregnant women with typical fetal CHD may play a key role in the development of CHD and may be used as novel biomarkers for prenatal diagnosis of fetal CHD.

MicroRNA-375 overexpression influences P19 cell proliferation, apoptosis and differentiation through the Notch signaling pathway.

Our previous study reported that microRNA-375 (miR-375) is significantly upregulated in ventricular septal myocardial tissues from 22­week­old fetuses with ventricular septal defect as compared with normal controls. In the present study, the specific effects of miR­375 on P19 cell differentiation into cardiomyocyte­like cells were investigated. Stable P19 cell lines overexpressing miR­375 or containing empty vector were established, which could be efficiently induced into cardiomyocyte­like cells in the presence of dimethyl sulfoxide in vitro. miR­375 overexpression was verified using reverse transcription­quantitative polymerase chain reaction (RT­qPCR). Cell proliferation was determined according to total cell counts; cell cycle distribution and apoptosis levels were examined using flow cytometry. Apoptosis­related morphological changes were observed using Hoechst staining and fluorescence microscopy. During P19 cell differentiation, the cardiomyogenesis­related mRNAs (cardiac troponin T, GATA binding protein 4, myocyte­specific enhancer factor 2C) and mRNAs involved in the Notch signaling pathway (Notch2, Delta­like 1 and hes family bHLH transcription factor 1) were detected at days 0, 4, 6 and 10. Their differential expression was examined using RT­qPCR; the apoptosis­related genes BAX and Bcl­2 were also detected using this method. The corresponding proteins were evaluated by western blotting. Compared with the control group, miR­375 overexpression inhibited proliferation but promoted apoptosis in P19 cells, and the associated mRNAs and proteins were decreased during differentiation. miR­375 has an important role in cardiomyocyte differentiation, and can disrupt this process via the Notch signaling pathway. The present findings contribute to the understanding of the mechanisms of congenital heart disease and facilitate the development of new gene therapies.

[Corrigendum] Effects of miR-19b knockdown on the cardiac differentiation of P19 mouse embryonic carcinoma cells.

Mol Med Rep 11: [Related article:] 2504­2512, 2015; DOI: 10.3892/mmr.2014.3037 Following the publication of this article, an interested reader drew to our attention some anomalies associated with the presentation of Fig. 7. In examining our original data, we identified that the upper and lower panels of 'D2' in Fig. 7A have been erroneously reproduced from the same image, which was derived from the negative control of the P19 cells ('vector'). A corrected version of the Figure is presented below, featuring the data which correctly correspond to day 2 ('D2') for the microRNA-19b knockdown experiment. We also checked the other figures very carefully, and failed to identify any further errors. We thank the reader of our article for drawing this matter to our attention, and we would like to offer our sincere apologies for this mistake, and our regrets for any inconvenience caused by this error.

Effect of miR-20b on Apoptosis, Differentiation, the BMP Signaling Pathway and Mitochondrial Function in the P19 Cell Model of Cardiac Differentiation In Vitro.

OBJECTIVE: To explore the effect of miR-20b on apoptosis, differentiation, the BMP signaling pathway and mitochondrial function in the P19 cell model of cardiac differentiation in vitro. METHODS: A miR-20b over-expression vector, a miR-20b silencing vector and their corresponding empty vectors were constructed and transfected into P19 cells, separately. Stably miR-20b overexpressing and silenced P19 cell lines were successfully selected by blasticidin and puromycin, separately. The cells were induced to undergo apoptosis in FBS-free-α-MEM. The induced cells were examined by flow cytometry and measurement of their caspase-3 activities. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to evaluate the relative expression of marker genes of cardiomyocytes during differentiation, such as cTnT, GATA4 and ANP. QRT-PCR was also used to detect the mitochondrial DNA (mtDNA) copy number. We investigated the cellular ATP production using a luciferase-based luminescence assay. The reactive oxygen species (ROS) was determined by DCFDA (2', 7'-Dichlorofluorescein diacetate) and the mitochondrial membrane potential (MMP) was elucidated by a JC-1 fluorescent probe, both using fluorescence microscopy and flow cytometer. The expression of BMP signaling pathway-related proteins were analyzed by Western blotting. RESULTS: Stably miR-20b overexpressing and silenced P19 cell lines were successfully obtained. MiR-20b overexpression increased apoptosis and promoted differentiation in P19 cells by promoting the activation of the BMP signaling pathway. In addition, miR-20b overexpression induced mitochondrial impairment in P19 cells during differentiation, which was characterized by lower MMP, raised ATP synthesis and increased ROS levels. The effects of miR-20b silencing were the exact opposite to those of overexpression. CONCLUSION: Collectively, these results suggested that miR-20b was very important in apoptosis, differentiation and mitochondrial function of P19 cells. MiR-20b may represent a new therapeutic target for congenital heart diseases and provide new insights into the mechanisms of cardiac diseases.