Fei Jiao

p53 Status and Its Prognostic Role in Extrahepatic Bile Duct Cancer: A Meta-Analysis of Published Studies

The dysfunction of p53 is the most common genetic alteration in human cancer. A variety of studies have investigated the clinicopathologic correlation of p53 and its impact on patient survival in different types of cancer. For extrahepatic bile duct cancer (EBDC), however, the results were limited and conflicting. In this study, we performed an investigation to confirm whether there was a correlation between p53 status and some routine parameters. To further observe the impact of p53 on the survival of EBDC patients, a meta-analysis based on published studies was conducted. Candidate studies were searched from PubMed, EMBASE, and ISI Web of Science. Our results demonstrated that there were significant correlations between p53 expression and some clinicopathological parameters. Furthermore, the pooled results of the meta-analysis showed that the combined hazard ratio (HR) estimate for overall survival (OS) was 1.53 (95% CI, 1.10–2.14) and 1.23 (95% CI, 0.93–1.75) in univariate and multivariate analysis, respectively. In conclusion, the high level of p53 appears to be an effective prognostic factor to OS of EBDC patients. However, some limitations unavoidable in this meta-analysis and problems of previous p53 studies in EBDC mean that further studies are necessary before significant conclusions can be made.

Protective effects of maternal methyl donor supplementation on adult offspring of high fat diet-fed dams

Obesity has become a global public health problem associated with metabolic dysfunction and chronic disorders. It has been shown that the risk of obesity and the DNA methylation profiles of the offspring can be affected by maternal nutrition, such as high-fat diet (HFD) consumption. The aim of this study was to investigate whether metabolic dysregulation and physiological abnormalities in offspring caused by maternal HFD can be alleviated by the treatment of methyl donors during pregnancy and lactation of dams. Female C57BL/6 mice were assigned to specific groups and given different nutrients (control diet, Control+Met, HFD and HFD+Met) throughout gestation and lactation. Offspring of each group were weaned onto a control diet at 3 weeks of age. Physiological (weight gain and adipose composition) and metabolic (plasma biochemical analyses) outcomes were assessed in male and female adult offspring. Expression and DNA methylation profiles of obesogenic-related genes including PPAR γ, fatty acid synthase, leptin and adiponectin were also detected in visceral fat of offspring. The results showed that dietary supplementation with methyl donors can prevent the adverse effects of maternal HFD on offspring. Changes in the expression and DNA methylation of obesogenic-related genes indicated that epigenetic regulation may contribute to the effects of maternal dietary factors on offspring outcomes.

DNA Methylation of Heparanase Promoter Influences Its Expression and Associated with the Progression of Human Breast Cancer

Heparanase promotes tumor invasion and metastasis in several malignancies including breast cancer. However, the roles and regulation mechanisms of heparanase during breast cancer progression are still not fully understood. The aim of this study is to determine the differential regulation of heparanase gene expression in specific stages of breast cancer by DNA methylation. We detected levels of heparanase expression and DNA methylation patterns of its promoter in breast cancer cell lines (MCF-7 and MDA-MB-435) and clinical tissues, respectively. It has been observed that heparanase is highly expressed in the invasive MDA-MB-435 cells with low methylation modification in the heparanase promoter. In contrast, lower expression of heparanase in MCF-7 cells is accompanied by higher methylation in the promoter. Treatment of MCF-7 cells with 5-aza-2′-deoxycytidine (5-aza-dC), a potent demethylating agent, results in induction of heparanase expression and higher invasion potential in vitro and leads to an advantage of tumor formation in vivo. In 54 tissue samples, cancer samples at late stages (stage IV) showed the highest heparanase expression accomplished by little DNA methylation. On the contrary, methylation prevalence is highest in normal tissue and inversely correlated with heparanase expression. A significant correlation between DNA methylation and clinical stage was demonstrated (p = 0.012). Collectively, these results demonstrate that DNA methylation play the regulation role in heparanase gene in different stages of breast cancer and present a direct effect on tumor progression.

Directed differentiation of chick embryonic germ cells into neural cells using retinoic acid induction in vitro

Embryonic germ cells (EG) are undifferentiated stem cells isolated from cultured primordial germ cells (PGC). Like embryonic stem cells (ES), EG are also capable of proliferation and self-renewal and have the capacity to differentiate in vitro into all cell types. To date, it has been proven that ES are capable of directed differentiation into neural precursors and progenies in mammals. However, similar studies on EG in mammals and other species are few. This investigation aimed to induce chick EG to differentiate into neural cells and compare the difference of efficiency between directed differentiation and spontaneous differentiation. EG were isolated and identified from 5.5-day chick gonadal PGC, incubated and passaged in conditioned medium. After the formation of embryoid bodies (EB), EB were grown in suspension and induced by retinoic acid (RA), using a protocol named 4-/4+, to make the formation of neurospheres and progenies. RT-PCR and immunocytochemistry analysis demonstrated that neural-specific markers can be detected after directed induction. Moreover, EG differentiated into neural lineage cells using 4-/4+ protocol much more efficiently than that in the spontaneous differentiation with fluorescence-activated cell sorting (FACS) analysis. The results revealed that RA can obviously promote the directed differentiation of chick EG into neural lineage.

Involvement of the mitochondrial pathway in p53-independent apoptosis induced by p28GANK knockdown in Hep3B cells.

It is well known that TP53 may mediate apoptosis triggered by anticancer drugs. However, accumulating evidence indicates that TP53 may be inactivated by mutations and/or deletions in about 50% of human cancers and, as such, may lead to pronounced resistance to therapeutic agents. Thus, the development of new approaches to improve the efficiency of therapeutic agents in cancer cells harboring mutant TP53 may have a significant impact on cancer treatment. It has been reported that knockdown by RNA interference (RNAi) of p28GANK (an alias of the gene PSMD10), a novel oncogene over-expressed in hepatocellular carcinoma (HCC), can induce apoptosis in HepG2, a TP53-intact HCC cell line. Because of the high frequency TP53 mutations in HCC, it is relevant to know whether p28GANK knockdown-induced apoptosis is also operational in TP53-negative HCC cells. Here, we investigated Adsip28GANK-induced apoptosis in the TP53-negative HCC cell line Hep3B. Our results indicate that p28GANK-knockdown induces the generation of reactive oxygen species (ROS), which in turn activates p38. Since p38 can signal to Bax, its activation may lead to mitochondrial transmembrane potential (Δψm) loss, cytochrome c release from mitochondria to cytosol, and caspase-9 activation, eventually triggering the mitochondrial pathway of apoptosis.

Maternal Chronic Folate Supplementation Ameliorates Behavior Disorders Induced by Prenatal High-Fat Diet Through Methylation Alteration of BDNF and Grin2b in Offspring Hippocampus

SCOPE Maternal consumption of a high-fat diet (HFD) during pregnancy increases the risk of behavioral problems. Folate plays an important role in neuroplasticity and the preservation of neuronal integrity. This study aims at determining the influence of diets supplemented with folate on offspring behavior, and the mechanisms involved. METHODS AND RESULTS Female mice were fed a control diet, an HFD, control diet supplemented with folate, or an HFD supplemented with folate for 5 weeks before mating. Open field task and elevated plus maze are used to evaluate the offspring behaviors. Results showed that offspring cognitive performance and anxiety-related behaviors, including those related to open field exploration and elevated plus maze, were significantly improved when dams were treated with folate in pregnancy. Moreover, the maternal folate supplement decreased BDNF and Grin2b methylation and upregulated their expressions in the brain of offspring, which were associated with decreasing the expression of DNA methyltransferases compared with those dams were treated only HFD in pregnancy. CONCLUSION Maternal folate supplementation ameliorates behavior disorders induced by prenatal high-fat diet. The beneficial effects were associated with methylation and expression alteration of BDNF and Grin2b genes.

Lipopolysaccharide Downregulates Kruppel-Like Factor 2 (KLF2) via Inducing DNMT1-Mediated Hypermethylation in Endothelial Cells

KLF2 plays a protective role in antiinflammation and endothelial function, and can be regulated by promoter methylation alteration. Lipopolysaccharide (LPS) is a mediator of inflammatory responses, which causes epigenetic change of certain genes in host cells. We thus aimed to determine whether LPS could control the KLF2 expression by inducing methylation in promoter region. DNA methylation of 16 CpG sites within KLF2 promoter region was detected by bisulfite sequencing PCR. Results showed that methylation at 12 CpG sites were significantly increased in HUVECs after exposure to LPS among the total 16 sites, and the average level was increased by 57%. The KLF2 expressions assessed by reverse transcription quantitative real-time PCR and Western blot were significantly downregulated compared that without LPS simulation. Moreover, both messenger RNA and protein levels of KLF2 in HUVEC co-treated with LPS and DNA methyltransferase (DNMT) 1 small interfering RNA were dramatically higher than that treated with LPS only. Similar result was obtained when the cells were incubated in combination with LPS and 5-aza-2′-deoxycytidine (AZA), suggesting that the reduction of KLF2 expression induced by LPS can be reversed by DNMT1 inhibition. Finally, the presence of AZA changed the expression of genes that depends on KLF2 in LPS-stimulated HUVECs, which downregulated the E-selectin and VCAM and increased the eNOS and thrombomodulin expression. Our data demonstrated that LPS exposure resulted in hypermethylation in KLF2 promoter in HUVECs, which subsequently led to downregulation of the KLF2 expression. The study suggested that epigenetic alteration is involved in LPS-induced inflammatory response and provided a new insight into atherogenesis.

Familial congenital cyanosis caused by Hb-M Yantai (α-76 GAC → TAC, Asp → Tyr)

Methemoglobin (Hb-M) is a rare hemoglobinopathy in China. We hereby report on a family living in Yantai, East China, with congenital cyanosis due to Hb-M mutation. The proband, a 65-year-old female, presented 63% oxygen saturation. Both Hb-M concentration and arterial oxygen saturation remained unchanged, even following intravenous treatment with methylene blue. There was also no change in blood-color (chocolate-brown) after adding 0.1% KCN. A fast-moving band (Hb-X) in hemolysates was found by cellulose acetate electrophoresis, the Hb-X/Hb-A ratio exceeding 10%. GT transition at 131nt of exon 2, although present in one of the α2 -globin alleles, was not found in α1 -globin alleles as a whole. This mutation leads to the aspartic acid to tyrosine substitution (Asp76Tyr). In this family, the novel mutation in the α2 -globin gene resulted in a rare form of congenital cyanosis due to Hb-M. This hemoglobin was named Hb-M Yantai .

Erythroid differentiation-related factor expression driven by effective promoter to ameliorate the anemia syndrome

The severity of β-thalassemia syndrome is associated with precipitation of the cytotoxic excessive free α-hemoglobin. We have established a therapy strategy aiming at stabilizing excessive free α-hemoglobin by decreasing its precipitation using erythroid differentiation-related factor (EDRF). To identify the functional EDRF promoter, different length of human EDRF promoter sequence was cloned upstream of green fluorescence protein (GFP) to drive GFP expression. After transfection, the intensity of GFP expression was monitored by microscopy and fluorescence activated cell sorter analysis. The 372 base pair sequence (−116∼+256 bp) was found to be most effective to induce the GFP expression and thereby cloned into pcDNA-EDRF vector to drive EDRF expression. After introducing pcDNA-EDRF vector into MEL cells and healthy mice, we confirmed high EDRF expression at both mRNA and protein levels using reverse transcription polymerase chain reacion and enzyme-linked immunosorbent assay. We further tested the function of EDRF overexpression in β-thalassemia mice by pcDNA-EDRF injection. One week later after treatment, the hemoglobin value, poikilocytosis and target cells of β-thalassemia mice were found to be ameliorated obviously. These results indicated that EDRF is a potential therapeutic target by improving hematological parameters of β-thalassemia.