Objective:

To investigate the incidence of aneuploidy in fetuses with congenital heart defects (CHDs) and to further identify submicroscopic changes and global DNA methylation levels as potential biomarkers in complex CHD cases.

Methods:

Fetuses at high risk for birth defects or with obvious sonographic anomalies were recruited at the Prenatal Diagnosis Center and Ultrasonic Diagnosis Center. Elective fetal karyotyping and DNA copy number and promoter methylation analyses were carried out following parental consent. G-banded karyotyping was performed to detect fetal aneuploidy. Copy number variations (CNVs) were detected using the Affymetrix SNP Array 6.0 and validated by real time PCR. Global DNA methylation analyses were conducted using a Roche NimbleGen Human DNA Methylation 3x720K Array, and DNA methylation differences were assayed by a Sequenom MassARRAY EpiTYPER.

Results:

Conventional karyotyping identified 30 cases with aneuploidy in 179 CHD fetuses. Various CNVs were found in two aneuploid fetuses and in five euploid CHD fetuses. Verified segmental deletion or duplications were not directly associated with cardiovascular malformations except in DAAM1 and GATA6. Verifiable aberrant DNA methylation could not be identified in three complex CHD fetuses.

Conclusions:

In this study, Trisomy 18, Trisomy 21 and 45,XO were the most common aneuploidies identified in CHD fetuses. In the affected samples, only DAAM1 deletion and GATA6 amplification could be associated with cardiovascular biological processes.

The present study was designed to investigate whether microRNAs (miRNAs) are involved in atrioventricular block (AVB) in the setting of myocardial ischemia (MI). A cardiac-specific miR-1 transgenic (Tg) mouse model was successfully established for the first time in this study using microinjection. miR-1 level was measured by real-time qRT-PCR. Whole-cell patch clamp was employed to record L-type calcium current (I Ca,L) and inward rectifier K(+) current (I K1). Expression of connexin 43 (Cx43) protein was determined by western blot analysis. Alternations of [Ca(2+)]i was detected by laser scanning confocal microscopy in ventricular myocytes. The incidence of AVB was higher in miR-1 Tg mice than that in wild-type (WT) mice. The normalized peak current amplitude of I Ca,L was lower in ventricular myocytes from miR-1 Tg mice as compared with WT mice. Similarly, the current density of I K1 was decreased in miR-1 Tg mice than that in WT mice. Compared with WT mice, miR-1 Tg mice exhibited a significant decrease of the systolic [Ca(2+)]i in ventricular myocytes but a prominent increase of the resting [Ca(2+)]i. Moreover, Cx43 protein was downregulated in miR-1 Tg mice compared to that in WT mice. Administration of LNA-modified antimiR-1 reversed all the above changes. miR-1 overexpression may contribute to the increased susceptibility of the heart to AVB, which provides us novel insights into the molecular mechanisms underlying ischemic cardiac arrhythmias.

Gene targeting (GT) by homologous recombination offers the best precision for genome editing in mice. nanos3 is a highly conserved gene and encodes a zinc-finger RNA binding protein essential for germ stem cell maintenance in Drosophila, zebrafish and mouse. Here we report nanos3 GT in embryonic stem (ES) cells of the fish medaka as a lower vertebrate model organism. A vector was designed for GT via homologous recombination on the basis of positive-negative selection (PNS). The ES cell line MES1 after gene transfer and PNS produced 56 colonies that were expanded into ES cell sublines. Nine sublines were GT-positive by PCR genotyping, 4 of which were homologous recombinants as revealed by Southern blot. We show that one of the 4, A15, contains a precisely targeted nanos3 allele without any random events, demonstrating the GT feasibility in medaka ES cells. Importantly, A15 retained all features of undifferentiated ES cells, including stable self-renewal, an undifferentiated phenotype, pluripotency gene expression and differentiation during chimeric embryogenesis. These results provide first evidence that the GT procedure and genuine GT on a chromosomal locus such as nanos3 do not compromise pluripotency in ES cells of a lower vertebrate.

As an important member of tyrosine kinase family, c-kit receptor causes specific expression of certain genes, regulates cell differentiation and proliferation, resists cell apoptosis, and plays a key role in tumor occurrence, development, migration and recurrence through activating the downstream signaling molecules following interaction with stem cell factor (SCF). The abnormality of SCF/c-kit signaling pathway is closely related to some certain tumors. The discovery of c-kit receptor-targeted drugs has promoted clinical-related cancer's diagnosis and treatment. In this paper, we review recent research progress on c-kit receptor-mediated signal transduction and its potential therapeutic application as a target in tumor-related diseases.

The p53-inducible gene 3 (PIG3) recently has been reported to be a new player in DNA damage signaling and response, but the crucial mechanism remains unclear. In the present study, the potential mechanism of PIG3 participation in the DNA damage response induced by ionizing radiation (IR) was investigated in multiple cell lines with depleted expression of PIG3 transiently or stably by the small interference RNA and lentivirus-mediated shRNA expression strategies. PIG3 knockdown led to an abnormal DNA damage response, including decreased IR-induced phosphorylation of H2AX, Chk1, Chk2 and Kap-1 as well as a prolonged G2-M arrest and aberrant mitotic progression. Notably, PIG3 knockdown resulted in a striking depression of cellular DNA-PKcs protein level, and was accompanied by a downregulation of ATM. Re-expression of PIG3 effectively rescued the depression of DNA-PKcs in PIG3-depleted cells. This negative regulation of DNA-PKcs by depleting PIG3 seemed to take place at the translational level but not at the levels of transcription or protein degradation. However, a compensatory feedback of increased mRNA expression of DNA-PKcs was formed in PIG3-depleted cells after a few passages or cell cycles of subculture, which led the recovery of the DNA-PKcs protein level and the consequent recovered efficiency of the DNA damage response. These results provide a new insight into the mechanism of PIG3's functioning in DNA damage signaling and the regulation network of cellular DNA-PKcs expression homeostasis.

The planted (l, d) motif search is one of the most widely studied problems in bioinformatics, which plays an important role in the identification of transcription factor binding sites in DNA sequences. However, it is still a challenging task to identify highly degenerate motifs, since current algorithms either output the exact results with a high computational cost or accomplish the computation in a short time but very often fall into a local optimum. In order to make a better trade-off between accuracy and efficiency, we propose a new pattern-driven algorithm, named PairMotif+. At first, some pairs of l-mers are extracted from input sequences according to probabilistic analysis and statistical method so that one or more pairs of motif instances are included in them. Then an approximate strategy for refining pairs of l-mers with high accuracy is adopted in order to avoid the verification of most candidate motifs. Experimental results on the simulated data show that PairMotif+ can solve various (l, d) problems within an hour on a PC with 2.67 GHz processor, and has a better identification accuracy than the compared algorithms MEME, AlignACE and VINE. Also, the validity of the proposed algorithm is tested on multiple real data sets.

Small synthetic compounds have been implicated in treatment of human cancers. We have synthesized a small compound, BPR1K0609S1 (hereafter, BP), which inhibits Aurora-A kinase. In the present study, we studied the mechanism of BP suppression of tumorigenesis induced by Aurora-A. Given our previous results that inactivation of p53 accelerates MMTV-Aurora-A-mediated tumorigenesis in vivo, we studied the roles of p53 pathway using the isogenic human colon carcinoma cell lines of HCT116, in which p53, Puma, Bax, p21 or Chk2 is deleted. When these isogenic cell lines are treated with BP for 48 h, accumulation of G2M phase and aneuploidy are commonly observed, and HCT116 p21(-) cells show increase in apoptosis. In xenograft assay, s.c. injection of BP efficiently inhibits tumorigenesis of HCT116 deficient for Chk2 or p21. Re-transplantation of BP-resistant tumors indicates that these resistant cells do not acquire advanced tumor growth. Significantly, 5-FU (5-fluorouracil) treatment further induces apoptosis of BP-resistant HCT116 deficient for Chk2 or Puma. These results demonstrate that p21 deficiency enhances BP-mediated suppression of tumor growth, and that BP and 5-FU can collaborate for tumor regression.

Myogenesis is a multistep process, in which myoblasts withdraw from the cell cycle, cease to divide, elongate and fuse to form multinucleated myotubes. Cell cycle transition is controlled by a family of cyclin-dependent protein kinases (CDKs) regulated by association with cyclins, negative regulatory subunits and phosphorylation. Muscle differentiation is orchestrated by myogenic regulatory factors (MRFs), such as MyoD and Myf-5. DNA methylation is crucial in transcriptional control of genes involved in myogenesis. Previous work has indicated that treatment of fibroblasts with the DNA-demethylating agent 5-azacytidine (AZA) promotes MyoD expression. We studied the effects of AZA on cell cycle regulation and MRFs synthesis during myoblast proliferation and early myogenesis phases in C2C12 cells. During the proliferation phase, cells were incubated in growth medium with 5µM AZA (GMAZA) or without AZA (GM) for 24 hours. At 70% confluence, cells were kept in growth medium in order to spontaneously achieve differentiation or transferred to differentiation medium with 5μM AZA (DMAZA) or without AZA (DM) for 12 and 24 hours. Cells used as control were unstimulated. In the proliferation phase, AZA-treated cells seemed to lose their characteristic circular shape and become elongated. The presence of AZA resulted in significant increases in the protein contents of Cyclin-D (FC:1.23 GMAZA vs GM p≤0.05), p21 (FC: 1.23 GMAZA vs GM p≤0.05), Myf-5 (FC: 1.21 GMAZA vs GM p≤0.05) and MyoD (FC: 1.20 GMAZA vs GM p≤0.05). These results propose that AZA could inhibit cell proliferation. During 12 hours of differentiation, AZA decreased the downregulation of genes involved in cell cycle arrest and in restriction point (G1 and G1/S phase) and the expression of several cyclins, E2F Transcription Factors, cyclin-dependent kinase inhibitors, specific genes responsible of cell cycle negative regulation. During 24 hours of differentiation, AZA induced an increment in the protein expression of Myf-5 (FC: 1.57 GMAZA vs GM p≤0.05), MyoD (FC: 1.14 DM vs GM p≤0.05; FC: 1.47 DMAZA vs GM p≤0.05), p21 (FC: 1.36 GMAZA vs GM p≤0.01; FC: 1.49 DM vs GM p≤0.05; FC: 1.82 DMAZA vs GM p≤0.01) and MyHC (FC: 1.40 GMAZA vs GM p≤0.01; FC: 2.39 DM vs GM p≤0.05; FC: 3.51 DMAZA vs GM p≤0.01). Our results suggest that AZA-induced DNA demethylation can modulate cell cycle progression and enhance myogenesis. The effects of AZA may open novel clinical uses in the field of muscle injury research and treatment.

Dihydropyrimidinase-related protein 4 (Dpysl4) is a known regulator of hippocampal neuron development. Here, we report that Dpysl4 is involved in growth regulation, polarization and differentiation of dental epithelial cells during tooth germ morphogenesis. A reduction in Dpysl4 gene expression in the tooth germ produced a loss of ameloblasts, resulting in the decrease of synthesis and secretion of enamel. The inhibition of Dpysl4 gene expression led to promotion of cell proliferation of inner enamel epithelial cells and inhibition of the differentiation of these cells into pre-ameloblasts, which was confirmed by analyzing cell polarization, columnar cell structure formation and the expression of ameloblast marker genes. By contrast, overexpression of Dpysl4 in dental epithelial cells induces inhibition of growth and increases the expression of the inner enamel epithelial cell marker gene, Msx2. These findings suggest that Dpysl4 plays essential roles in tooth germ morphogenesis through the regulation of dental epithelial cell proliferation, cell polarization and differentiation.

RNA interference (RNAi) caused by exogenous double-stranded RNA (dsRNA) has developed into a powerful technique in functional genomics, and to date it is widely used to down-regulate crucial physiology-related genes to control pest insects. A molt-regulating transcription factor gene, HaHR3, of cotton bollworm (Helicoverpa armigera) was selected as the target gene. Four different fragments covering the coding sequence (CDS) of HaHR3 were cloned into vector L4440 to express dsRNAs in Escherichia coli. The most effective silencing fragment was then cloned into a plant over-expression vector to express a hairpin RNA (hpRNA) in transgenic tobacco (Nicotiana tabacum). When H. armigera larvae were fed the E. coli or transgenic plants, the HaHR3 mRNA and protein levels dramatically decreased, resulting developmental deformity and larval lethality. The results demonstrate that both recombinant bacteria and transgenic plants could induce HaHR3 silence to disrupt H. armigera development, transgenic plant-mediated RNAi is emerging as a powerful approach for controlling insect pests.