RETRACTION: "Mechanical stretch promotes antioxidant responses and cardiomyogenic differentiation in P19 cells".

Objective To explore the effects of microRNA(miRNA)-30c knockdown on proliferation, diffe-rentiation of P19 cells. Methods miRNA-30c knockdown plasmid(miRNA-30c knockdown group) or no-load vector(negative control group) was transfected into P19 cells by lipo2000 and stable cell lines were selected by Blasticidin; Dual luciferase reporter gene system was used to confirm miRNA-30c knockdown.Cell counting kit-8(CCK-8) assay was adopted to detect cell proliferation activity.An inverted microscope was used to observe morphological changes of P19 cell differentiation.Cells were induced to differentiated to myocardiocyte with dimethyl sulfoxide(DMSO). Differentiation marker genes including cTnT, NKX2.5, GATA4 relative mRNA expression levels were detected with real-time quantitative polymerase chain reaction, respectively. Results Observation of green fluorescent protein expression under a fluorescence microscope indicated similar transfection efficiencies, and miRNA-30c knockdown released the activity of target gene Gli2.As a result, miRNA-30c knockdown vector was constructed successfully(P<0.001). During differentiation of mouse P19 cells into myocardial cells, the beating cell clusters in miRNA-30c knockdown cells were much lower than those in the control cells, and cTnT, NKX2.5, GATA4 in miRNA-30c knockdown cells showed significantly lower expression than those in the control cells(all P<0.05). Conclusions miRNA-30c inhibits the P19 cell proliferation and differentiation.This study gives us a new insight of heart development and we need more efforts on exploring the deep function of heart diseases. Key words: microRNA-30c; P19 cell; Proliferation; Differentiation

In this study, we describe a role for the mammalian Numb-interacting protein 1 (Nip1) in regulation of neuronal differentiation in stem cells. The expression of Nip1 was detected in the developing mouse brain, embryonic stem cells, primary neuronal stem cells, and retinoic acid-treated P19 embryonal carcinoma cells. The highest expression of Nip1 was observed in undifferentiated neuronal stem cells and was associated with Duox1-mediated reactive oxygen species ROS production. Ectopic nip1 expression in P19 embryonal carcinoma cells induced neuronal differentiation, and this phenotype was also linked to elevated ROS production. The neuronal differentiation in nip1-overexpressing P19 cells was achieved in a retinoic acid-independent manner and was corroborated by an increase in the expression of the neuronal basic helix-loop-helix transcription factors and neural-lineage cell markers. Furthermore, depletion of nip1 by short hairpin RNA led to a decrease in the expression of neuronal basic helix-loop-helix transcription factors and ROS. However, inhibition of ROS production in nip1-overexpressing P19 cells restricted but did not extinguish neuronal differentiation. Microarray and mass spectrometry analysis identified intermediate filaments as the principal cytoskeletal elements affected by up-regulation of nip1. We show here the first evidence for a functional interaction between Nip1 and a component of the nuclear lamina, lamin A/C. associated with a neuronal-specific phenotype. Taken together, our data reveal an important role for Nip1 in the guidance of neuronal differentiation through ROS generation and modulation of intermediate filaments and implicate Nip1 as a novel intrinsic regulator of neuronal cell fate.

Directed differentiation of embryonic P19 cells and neural stem cells into neural lineage on conducting PEDOT–PEG and ITO glass substrates

Kinins are vasoactive oligopeptides generated upon proteolytic cleavage of low and high molecular weight kininogens by kallikreins. These peptides have a well established signaling role in inflammation and homeostasis. Nevertheless, emerging evidence suggests that bradykinin and other kinins are stored in the central nervous system and may act as neuromediators in the control of nociceptive response. Here we show that the kinin-B2 receptor (B2BKR) is differentially expressed during in vitro neuronal differentiation of P19 cells. Following induction by retinoic acid, cells form embryonic bodies and then undergo neuronal differentiation, which is complete after 8 and 9 days. Immunochemical staining revealed that B2BKR protein expression was below detection limits in nondifferentiated P19 cells but increased during the course of neuronal differentiation and peaked on days 8 and 9. Measurement of [Ca(2+)](i) in the absence and presence of bradykinin showed that most undifferentiated cells are unresponsive to bradykinin application, but following differentiation, P19 cells express high molecular weight neurofilaments, secrete bradykinin into the culture medium, and respond to bradykinin application with a transient increase in [Ca(2+)](i). However, inhibition of B2BKR activity with HOE-140 during early differentiation led to a decrease in the size of embryonic bodies formed. Pretreatment of differentiating P19 cells with HOE-140 on day 5 resulted in a reduction of the calcium response induced by the cholinergic agonist carbamoylcholine and decreased expression levels of M1-M3 muscarinic acetylcholine receptors, indicating crucial functions of the B2BKR during neuronal differentiation.

ABSTRACTMicroRNA (miR)-20a, a member of the miR-17-92 cluster related to cardiac development, was obviously downregulated in myocardially differentiated P19 cells compared with normal P19 cells. Smoothened (SMO) is a member of the Hh pathway. Hh signaling induces cardiac differentiation in P19 cells, and SMO mediates the Hh pathway during embryonic development. Using bioinformatic prediction software Targetscan (http://www.targetscan.org/), PicTar (http://pictar.bio.nyu.edu), and miRBase (http://microrna.sanger.ac.uk/), miR-20a and the 3′-untranslated region (3′-UTR) of SMO mRNA were predicted to have complementary binding regions. Accordingly, we inferred that miR-20a might act as a regulator of SMO, and regulate proliferation, differentiation and apoptosis in P19 cells. We determined the expression of miR-20a, SMO and marker proteins of cardiomyocytes (cTnT, GATA4 and desmin) by quantitative real-time PCR (qRT-PCR) and western blot assays, and found that P19 cells had differentiated into cardiomyocytes successfully at differentiation day 10, and downregulation of miR-20a and upregulation of SMO existed in myocardially differentiated P19 cells. Cell proliferation, differentiation and apoptosis detection showed that miR-20a upregulation inhibited proliferation and differentiation and enhanced apoptosis in P19 cells. Moreover, we verified that miR-20a directly targeted SMO and knockdown of SMO and miR-20a overexpression had similar effects on P19 cell proliferation, differentiation and apoptosis, which verified the speculation that miR-20a inhibits proliferation and differentiation and enhances apoptosis in P19 cells by directly targeting SMO. Our results suggest that miR-20a may be a potential target against congenital heart diseases.

Our previous study reported that microRNA-375 (miR-375) is significantly upregulated in ventricular septal myocardial tissues from 22-week-old fetuses with ventricular septal defect as compared with normal controls. In the present study, the specific effects of miR-375 on P19 cell differentiation into cardiomyocyte-like cells were investigated. Stable P19 cell lines overexpressing miR-375 or containing empty vector were established, which could be efficiently induced into cardiomyocyte-like cells in the presence of dimethyl sulfoxide in vitro. miR-375 overexpression was verified using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cell proliferation was determined according to total cell counts; cell cycle distribution and apoptosis levels were examined using flow cytometry. Apoptosis-related morphological changes were observed using Hoechst staining and fluorescence microscopy. During P19 cell differentiation, the cardiomyogenesis-related mRNAs (cardiac troponin T, GATA binding protein 4, myocyte-specific enhancer factor 2C) and mRNAs involved in the Notch signaling pathway (Notch2, Delta-like 1 and hes family bHLH transcription factor 1) were detected at days 0, 4, 6 and 10. Their differential expression was examined using RT-qPCR; the apoptosis-related genes BAX and Bcl-2 were also detected using this method. The corresponding proteins were evaluated by western blotting. Compared with the control group, miR-375 overexpression inhibited proliferation but promoted apoptosis in P19 cells, and the associated mRNAs and proteins were decreased during differentiation. miR-375 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.

Accumulating evidence has suggested that mechanical stimuli play a crucial role in regulating the lineage-specific differentiation of stem cells through fine-tuning redox balance. We aimed to investigate the effects of cyclic tensile strain (CTS) on the expression of antioxidant enzymes and cardiac-specific genes in P19 cells, a widely characterized tool for cardiac differentiation research. A stretching device was applied to generate different magnitude and duration of cyclic strains on P19 cells. The messenger RNA and protein levels of targeted genes were determined by real-time polymerase chain reaction and Western blot assays, respectively. Proper magnitude and duration of cognitivestimulationtherapy (CST) stimulation substantially enhanced the expression of both antioxidant enzymes and cardiac-specific genes in P19 cells. Sirtuin 1 (SIRT1) played an essential role in the CTS-induced cardiomyogenic differentiation of P19, as evidenced by changes in the expression of antioxidant enzymes and cardiac-specific genes. Mechanical loading promoted the cardiomyogenic differentiation of P19 cells. SIRT1 was involved in CST-mediated P19 differentiation, implying that SIRT1 might serve as an important target for developing methods to promote cardiomyogenic differentiation of stem cells.

The molecular mechanisms involved in neuronal/astroglial cell fate decisions during the development of the mammalian central nervous system are poorly understood. Here, we report that PRP19beta, a splice variant of mouse PRP19alpha corresponding to the yeast PRP19 protein, can function as a neuron-astroglial switch during the retinoic acid-primed neural differentiation of P19 cells. The beta-variant possesses an additional 19 amino acid residues in-frame in the N-terminal region of the alpha-variant. The forced expression of the alpha-variant RNA caused the down-regulation of oct-3/4 and nanog mRNA expression during the 12-48 h of the late-early stages of neural differentiation and was sufficient to convert P19 cells into neurons (but not glial cells) when the cells were cultured in aggregated form without retinoic acid. In contrast, the forced expression of the beta-variant RNA suppressed neuronal differentiation and conversely stimulated astroglial cell differentiation in retinoic acid-primed P19 cells. Based on yeast two-hybrid screening, cyclophilin A was identified as a specific binding partner of the beta-variant. Luciferase reporter assay mediated by the oct-3/4 promoter revealed that cyclophilin A could act as a transcriptional activator and that its activity was suppressed by the beta-variant, suggesting that cyclophilin A takes part in the induction of oct-3/4 gene expression, which might lead to neuroectodermal otx2 expression within 12 h of the immediate-early stages of retinoic acid-primed neural differentiation. These results show that the alpha-variant gene plays a pivotal role in neural differentiation and that the beta-variant participates in neuronal/astroglial cell fate decisions.

T-cell acute lymphocytic leukemia 2 (Tal2) is a gene encoding a member of the basic helix-loop-helix transcription factor family, which is essential for the normal development of the mouse brain. We found that Tal2 was induced during neural differentiation in P19 cells, which are pluripotent mouse embryonal carcinoma cells that differentiate into the neural lineage upon both exposure to all-trans retinoic acid (atRA) and the formation of cell aggregation. Tal2 expression during neural differentiation in P19 cells was detected within 3 h after induction with atRA and retinoic acid receptor α (RARα). The atRA-RARα complex is known to bind to a characteristic retinoic acid response element (RARE) located in the promoter of target genes. We found a RARE-like element in the intron of Tal2. We also found a TATA-box-like element in the 5' region. The TATA-box-like element functioned as a core promoter, and TATA- box binding protein bound to this element upstream of Tal2 in P19 cells. The RARE-like element responded to atRA signaling that activated the transcription, and RARα was bound to this element in the intron of Tal2 in P19 cells. Furthermore, the interaction between these elements on Tal2 was confirmed in a chromatin immunoprecipitation assay. Because the neural differentiation of P19 cells mimics in part the development of the nervous system, P19 cells are useful for studying the mechanism underlying the role of Tal2 in neural differentiation. Further work is underway to clarify the function of Tal2 in neural differentiation using the differentiation system of P19 cells.

Stable transfectants with expression of small interfering RNA for targeting cyclophilin A (CypA) in p19 cells lose their potential for retinoic acid (RA)-induced neuronal differentiation but not Me(2)SO-induced mesodermal differentiation. This difference suggests that CypA is specifically required for the RA-induced neuronal pathway. In addition to the loss of RA-induced RA receptor beta expression and retinoic acid response element (RARE)-binding activity, a dramatic reduction in RA-induced RARE-mediated luciferase activity in the CypA knockdown cell line suggests that CypA affects RARE-mediated regulation of gene expression. Silent mutation of target sequences confirms the specificity of RNA interference in p19 embryonal carcinoma cells. Collectively, our data reveal that a novel function of CypA is required in the processing of RA-induced neuronal differentiation in p19 embryonal carcinoma cells.

Objective To observe the expression changes in microRNA(miR)-379 in the developmental process of the mouse heart and during the differentiation of P19 cells into cardiac myocytes, and to explore the possible relationship between miR-379 and the differentiation of cardiacmyocytes. Methods Heart tissues were collected from fetal mice in pregnant ones at their gestational age (8.5, 11.5, 14.5 and 18.5 days) respectively.Heart tissue sections of the fatal mice were obtained to observe the heart development process.Then total RNA was isolated from heart tissues by using the TRIzol method.Complementary DNA was synthesized from 1 μg total RNA by using a Reverse Transcriptase Kit.Finally, real-time PCR (RT-PCR) was employed to detect the expression of miR-379.At the same time, P19 cells were cultured with 10 mL/L Dimethyl sulfoxide in suspension for 4 days to form cell aggregation, and these aggregations were transferred into 6-wells plate for culturing by adherence.Beating cells were detected with microscopy on the 10th day after induction.Afterwards, total RNA was extracted from cultured P19 cells at different time points.Reverse transcription was executed to get DNA.At last, RT-PCR was used to explore the expression of miR-379 on 0, 4, 6, 10 days after aggregation. Results The expression level of miR-379 was down-regulated gradually in the developing heart(at gestational age of 8.5, 11.5, 14.5, 16.5 days, respectively), and there were significant differences on the different days(F=21.13, P<0.05). On the other hand, myocardial markers of troponin T represented an increasing trend during the process of P19 cells induction, which demonstrated that P19 cells were successfully induced into cardiomyocyte-like cells.Meanwhile, miR-379 showed a low expression on day 0 of P19 cells aggregation.On day 4, miR-379 demonstrated a higher level.Afterwards, miR-379 proved to be down-regulated gradually. Conclusions miR-379 plays a role in the process of the heart development, but the specific mechanisms need further research. Key words: MicroRNA-379; Cardiac differentiation; Heart tissue; P19 cell

Sox6 is a transcription factor that induces neuronal differentiation in P19 cells; its suppression not only inhibits neuronal differentiation but also induces retinoic acid (RA)-dependent apoptosis of P19 cells. In the present study, we found that Sox6 suppression-induced apoptosis was mediated by activation of caspase 9 and 3. Moreover, we noted a weak leakage of cytochrome c into the cytoplasm from the mitochondria, indicating that apoptosis occurs through a mitochondrial pathway in Sox6-suppressed P19 (P19[anti-Sox6]) cells. Sox6 suppression in the presence of RA also induced the expression and secretion of bone morphogenetic protein 4 (BMP-4). Addition of an anti-BMP-4 antibody for neutralization increased cell viability and led to RA-dependent death of P19[anti-Sox6] cells. Our results indicate that Sox6 suppression induces RA-dependent cell death of P19 cells, mediated by BMP-4 expression and secretion. Normally, high Sox6 expression leads to RA-mediated neuronal differentiation in P19 cells; however, Sox6 deficiency induces production and secretion of BMP-4, which mediates selective cell death. Our findings suggest that Sox6 contributes to cell survival by suppressing BMP-4 transcription during neuronal differentiation.

Cyclin-dependent kinase 5 (cdk5)/p35 kinase activity is highest in post-mitotic neurons of the central nervous system and is critical for development and function of the brain. The neuronal specific activity of the cdk5/p35 kinase is achieved through the regulated expression of p35 mRNA. We have identified a small 200-bp fragment of the p35 promoter that is sufficient for high levels of neuronal specific expression. Mutational analysis of this TATA-less promoter has identified a 17-bp GC-rich element, present twice, that is both required for promoter activity and sufficient for neuronal specific transcription. A GC box within the 17-bp element is critical for both promoter activity and protein-DNA complex formation. The related transcription factors Sp1, Sp3, and Sp4 constitute most of the GC box DNA binding activity in neurons. We have found that both the relative contribution of the Sp family proteins to GC box binding and the transcriptional activity of these proteins is regulated during neuronal differentiation. Thus, our data show that the GC box-binding Sp proteins contribute to the regulation of p35 expression in neurons, suggesting changes in the Sp transcription factors level and activity may contribute to cell type-specific expression of many genes in the central nervous system.

A Role of N-Cadherin in Neuronal Differentiation of Embryonic Carcinoma P19 Cells

This study aimed to investigate the role and regulatory mechanism of small nucleolar RNA host gene 6 (SNHG6), a long noncoding RNA, in the formation of ventricular septal defect (VSD). The expression of SNHG6 in fetal cardiac tissues with VSD, mouse heart embryo development and the differentiation of P19 cells into cardiomyocytes were determined. Moreover, the effect of aberrant expression of SNHG6 on P19 cell proliferation, cell cycle, apoptosis and differentiation was further analyzed to explore the role of SNHG6 in affecting myocardial development. Furthermore, the regulatory mechanism between SNHG6 and miR-101 as well as between SNHG6 and activation of Wnt/β-catenin pathway was investigated. SNHG6 was upregulated in fetal cardiac tissues with VSD, and decreased in the embryonic development of mice and differentiation of P19 cells into cardiomyocytes. Overexpression of SNHG6 inhibited P19 cell proliferation and induced apoptosis, as well as promoted cell differentiation into cardiomyocytes. Furthermore, SNHG6 could negative regulate the expression of miR-101, and the effects of SNHG6 on the modulation of P19 cell function were through negative regulation of miR-101. In addition, overexpression of SNHG6 activated Wnt/β-catenin pathway, which was reversed after overexpression of SNHG6 and miR-101 synchronously. Our study reveals that SNHG6 may contribute to VSD formation via negative regulation of miR-101 and activation of Wnt/β-catenin pathway. SNHG6 may constitute a potential therapeutic target in this disease.