Effect of LYRM1 knockdown on proliferation, apoptosis, differentiation and mitochondrial function in the P19 cell model of cardiac differentiation in vitro.

To explore the effects of LYRM1 knockdown on proliferation, apoptosis, differentiation and mitochondrial function in the embryonic carcinoma (P19) cell model of cardiac differentiation. Knockdown of LYRM1 using small interfering RNA (siRNA) was confirmed by quantitative real-time PCR. Cell Counting Kit-8(CCK-8) proliferation assays and cell cycle analysis demonstrated that LYRM1 gene silencing significantly inhibited P19 cell proliferation. Flow cytometry and measurement of their caspase-3 activities revealed that knockdown of LYRM1 increased P19 cell apoptosis. Observation of morphological changes using an inverted microscope and expression analysis of specific differentiation marker genes using quantitative real-time PCR and Western blotting revealed that knockdown of LYRM1 significantly inhibited the differentiation of P19 cells into cardiomyocytes. Furthermore, real-time quantitative PCR applied to detect mitochondrial DNA (mtDNA) copy number implied that there was no significant difference in the LYRM1 knockdown group compared with the control group. Cellular ATP production investigated by luciferase-based luminescence assay was dramatically decreased in differentiated cells transfected with LYRM1 RNAi. Fluorescence microscopy and flow cytometery were used to detect the reactive oxygen species (ROS) and the mitochondrial membrane potential (MMP) showed that the level of ROS was dramatically increased and MMP was obviously decreased in differentiated cells transfected with LYRM1 RNAi. Collectively, knockdown of LYRM1 promoted apoptosis and suppressed proliferation and differentiation in P19 cells. In addition, knockdown of LYRM1 induced mitochondrial impairment in P19 cells during differentiation, which was reflected by decreased ATP synthesis, lower MMP and increased ROS levels.

Overexpression of FABP3 promotes apoptosis through inducing mitochondrial impairment in embryonic cancer cells.

Fatty acid-binding protein 3 (FABP3) is a low-molecular-weight protein with a distinct tissue distribution that may play an important role in fatty acid transport, cell growth, cellular signaling, and gene transcription. Previously, we have found that FABP3 was involved in apoptosis-associated congenital cardiac malformations, but the underlying mechanisms have not yet been described. In the present study, we investigated the characteristics of mitochondrial dysfunction in embryonic cancer cells (P19 cells) that overexpressed FABP3. We demonstrated that in FABP3-overexpressing P19 cells a lower cellular ATP production was accompanied by a dramatic decrease in mitochondrial membrane potential (MMP), despite the lack of a substantial decrease in the mtDNA copy number. In addition, FABP3 overexpression also led to an imbalance in mitochondrial dynamics and to excess intracellular reactive oxygen species production. Collectively, our results indicated that overexpression of FABP3 in P19 cells caused mitochondrion dysfunction that might be responsible for the development of FABP3-induced apoptosis.

Silencing of FABP3 promotes apoptosis and induces mitochondrion impairment in embryonic carcinoma cells.

Fatty acid binding protein 3 (FABP3) (also known as H-FABP) is a member of the intracellular lipid-binding protein family, and is mainly expressed in cardiac muscle tissue. The in vivo function of FABP3 is proposed to be in fatty acid metabolism, trafficking, and cell signaling. Our previous study found that FABP3 is highly regulated in patients with ventricular septal defect (VSD), and may play a significant role in the development of human VSD. In the present study, we aimed to investigate the impact of FABP3 knockdown by RNA interference (RNAi) on apoptosis and mitochondrial function of embryonic carcinoma (P19) cells. The results revealed that downregulated FABP3 expression promoted apoptosis, and resulted in mitochondrial deformation, increased mitochondrial membrane potential (MMP), and decreased intracellular ATP synthesis. In addition, the knockdown of FABP3 also led to excess intracellular ROS production. However, there was no obvious influence on the amount of mitochondrial DNA. Collectively, our results indicated that FABP3 knockdown promoted apoptosis and caused mitochondrial dysfunction in P19 cells, which might be responsible for the development of human VSD.

LYRM1, a gene that promotes proliferation and inhibits apoptosis during heart development.

Congenital heart disease (CHD) is the most common type of birth defect, but its underlying molecular mechanisms remain unidentified. Previous studies determined that Homo sapiens LYR motif containing 1 (LYRM1) is a novel nucleoprotein expressed at the highest level in adipose tissue and in high levels in heart tissue. The LYRM1 gene may play an important role in the development of the human heart. This study was designed to identify the biological characteristics of the LYRM1 gene in heart development. On the basis of expression-specific differentiation markers identified with quantitative real-time RT-PCR and the morphology of LYRM1-overexpressing cells during differentiation, ectopic expression was not found to significantly affect differentiation of P19 cells into cardiomyocytes. MTT assays and cell cycle analysis showed that LYRM1 dramatically increases the proliferation of P19 cells. Furthermore, data from annexin V-FITC binding and caspase-3 activity revealed that LYRM1 can inhibit the apoptosis of P19 cells. Our data suggest that LYRM1 might have the potential to modulate cell growth, apoptosis, and heart development.

Pancreatic carcinoma underlying a complex presentation in late pregnancy: a case report.

BACKGROUND: Gestational diabetes mellitus is strongly related to the risk of pancreatic cancer in pregnant women, but gestational diabetes can precede a diagnosis of pancreatic cancer by many years. Women with a history of gestational diabetes showed a relative risk of pancreatic cancer of 7.1. Pancreatic adenocarcinoma is one of the most common malignancies associated with thromboembolic events. A clinical study showed that thromboembolic events were detected in 36% of patients diagnosed as having pancreatic cancer. Studies showed that gestational diabetes mellitus could be one of the important risk factors for pancreatic cancer. CASE PRESENTATION: Gestational diabetes mellitus is associated with increased risk of breast and pancreatic cancer. This case report describes a 29-year-old Chinese woman who presented with: gestational diabetes mellitus; International Society on Thrombosis and Haemostasis criteria suggested disseminated intravascular coagulation with a score of 5; hemolysis, elevated liver enzymes, low platelet count syndrome; and pulmonary hypertension. After an intravenous injection of fibrinogen, she gave birth to a normal baby and following delivery, her blood pressure reached 180/110 mmHg. Laboratory analysis results showed elevated lactic dehydrogenase, decreased platelets and fibrinogen, and urine protein was positive. She was transfused with fresh frozen plasma, blood coagulation factor, and fibrinogen. Subsequently, she was transferred to a maternity intensive care unit, where magnesium sulfate seizure prophylaxis was continued for 24 hours to keep her magnesium level at a low therapeutic range. However, continuous oxygen therapy was needed to maintain her oxygenation. Further laboratory investigations revealed elevated carcinoembryonic antigen, carbohydrate antigen 19-9, and carbohydrate antigen 72-4. Positron emission tomography-computed tomography showed malignant carcinoma in the head of her pancreas with lymph node involvement along with bone, peritoneal, and left adrenal metastasis, as well as double lung lymphangitic carcinomatosis. CONCLUSION: A differential diagnosis of digestive system neoplasm should be considered when a pregnant patient presents with gestational diabetes mellitus and disseminated intravascular coagulation, where the disseminated intravascular coagulation has no specific cause and cannot be readily resolved.

Identification of differentially expressed genes involved in transient regeneration of the neonatal C57BL/6J mouse heart by digital gene expression profiling.

Accumulating evidence has revealed that the mammalian heart possesses a measurable capacity for renewal. Neonatal mice retain a regenerative capacity over a short time-frame (≤6 days), but this capacity is lost by 7 days of age. In the present study, differential gene expression profiling of mouse cardiac tissue was performed to further elucidate the mechanisms underlying this process. The global gene expression patterns of the neonatal C57BL/6J mouse heart were examined at three key time-points (1, 6 and 7 days old) using digital gene expression analysis. In the distribution of total clean tags, high-expression tags (>100 copies) were found to be predominant, whereas low expression tags (<5 copies) occupied the majority of distinct tag distributions. In total, 306 differentially expressed genes (DEGs) were detected in cardiac tissue, with the expression levels of 115 genes upregulated and those of 191 genes downregulated in 7-day-old mice compared with expression levels in 1- and 6-day-old mice, respectively. The expression levels of five DEGs were confirmed using quantitative polymerase chain reaction. Gene ontology analysis revealed a large proportion of DEGs distributed throughout the cell, and these DEGs were associated with binding as well as catalytic, hydrolase, transferase and molecular transducer activities. Furthermore, these genes were involved in cellular, metabolic and developmental processes, as well as biological regulation and signaling pathways. Pathway analysis identified the oxidative phosphorylation pathway to be the process most significantly putatively affected by the differential expression of these genes. These data provide the basis for future analysis of the gene expression patterns that regulate the molecular mechanism of cardiac regeneration.

Silencing of FABP3 leads to apoptosis-induced mitochondrial dysfunction and stimulates Wnt signaling in zebrafish.

Fatty acid binding protein 3 (FABP3, also termed heart-type fatty acid binding protein) is a member of the intracellular lipid-binding protein family that may be essential in fatty acid transport, cell growth, cellular signaling and gene transcription. Previously, we demonstrated that FABP3 was involved in apoptosis-associated congenital cardiac malformations; however, its mechanism of regulation remains unclear. Apoptosis has increasingly been considered to be important in cardiac development. In the present study, a zebrafish model was used to investigate the involvement of FABP3­morpholino (MO)-induced apoptosis and mitochondrial dysfunction in cardiac development. During the early stages of cardiac development, injection of FABP3­MO into zebrafish resulted in significant impairment in cardiac development and promoted the rate of apoptosis which was correlated with significant dysfunction of the mitochondria. For example, the ATP content was markedly decreased at 24 and 48 h post-fertilization (pf), reactive oxygen species production was significantly enhanced at 24 and 48 h pf and the mitochondrial DNA copy number was reduced at 24, 48 and 72 h pf. Additionally, Nkx2.5 expression was upregulated in FABP3-MO zebrafish, and Wnt signaling molecules (Wnt1, Wnt5 and Wnt11) were also highly expressed in FABP3-MO zebrafish at 24, 48 and 72 h pf. In conclusion, the results indicated that FABP3 knockdown exhibited significant toxic effects on cardiac development and mitochondrial function, which may be responsible for the knockdown of FABP3-induced apoptosis. Apoptosis was one of the mechanisms underlying this effect, and was correlated with the activation of Wnt signaling. These studies identified FABP3 as a candidate gene underlying the etiology of congenital heart defects.

Differential expression of microRNAs in cardiac myocytes compared to undifferentiated P19 cells.

microRNA (miRNA) expression is tightly controlled in a tissue-specific and developmental stage-specific manner; some are highly and specifically expressed in cardiovascular tissues. miRNA expression profiling, using miRNA microarrays facilitates studying the biological function of miRNAs. We investigated changes in miRNA expression profiles during differentiation of P19 cells into cardiac myocytes in order to elucidate the mechanisms of heart development. The morphology of P19 cells during differentiation was observed using an inverted microscope. Western blot analysis was performed to detect cardiac troponin I (cTnI) expression. Total RNA was extracted from P19 cells for microarray and real-time quantitative reverse transcription-polymerase chain reaction (real-time qRT-PCR) analyses to determine the miRNA expression profile. The miRNA microarray revealed differential expression of 49 miRNAs, of which 26 were down-regulated and 23 were up-regulated in differentiated cardiac myocytes, compared to normal P19 cells. This was confirmed by real-time qRT-PCR. We also utilized target prediction analysis to identify gene targets. Some miRNAs may have important roles in cardiac development and congenital heart defects (CHDs). Further analysis of miRNA function to confirm their target genes during cardiac development will determine the potential for novel miRNA-based therapeutic strategies.

GATA-4 promotes the differentiation of P19 cells into cardiac myocytes.

The aim of this study was to investigate the effects of GATA-4 on the differentiation of P19 cells into cardiomyocytes and to examine the relationship between GATA-4 and cardiomyocytes. We constructed vectors to overexpress and silence GATA-4. These vectors, as well as empty ones were transfected into P19 cells. Subsequently, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis were performed. The morphology of P19 cells during differentiation was observed using an inverted microscope. Total RNA was extracted from P19 cells. We used real-time PCR to evaluate the expression levels of 6 genes: GATA-4, GATA-6, transthyretin (TTR), alpha-fetoprotein (AFP), Nkx2.5, and alpha-myosin heavy chain (alpha-MHC). The gene expression pattern of these 6 genes is graphically shown for each group. The GATA-4 mRNA level in cells overexpressing GATA-4 was notably higher than that in the controls, whereas the levels in the controls were notably higher than those in the GATA-4-silenced P19 cells. The cell lines overexpressing GATA-4 expressed higher levels of Nkx2.5 and alpha-MHC than the controls. However, the controls expressed higher levels of AFP, GATA-6 and TTR than the cells overexpressing GATA-4. The RNAi group expressed lower levels of TTR, Nkx2.5, and alpha-MHC than the controls, but there were no differences in the RNAi group and the controls with regard to the expression levels of AFP and GATA-6. The gene expression pattern in the cells overexpressing GATA-4 was biased toward the Nkx2.5 and alpha-MHC. On the other hand, the gene expression pattern in GATA-4-silenced cells and the controls was biased toward the TTR and AFP. The overexpression of GATA-4 enhances the differentiation of P19 cells into cardiac myocytes, whereas its down-regulation suppresses this trend.