BACKGROUND: Prenatal stress can cause neurobiological and behavioral defects in offspring; environmental factors play a crucial role in regulating the development of brain and behavioral; this study was designed to test and verify whether an enriched environment can repair learning and memory impairment in offspring rats induced by prenatal stress and to explore its mechanism involving the expression of insulin-like growth factor-2 (IGF-2) and activity-regulated cytoskeletal-associated protein (Arc) in the hippocampus of the offspring. METHODS: Rats were selected to establish a chronic unpredictable mild stress (CUMS) model during pregnancy. Offspring were weaned on 21st day and housed under either standard or an enriched environment. The learning and memory ability were tested using Morris water maze and Y-maze. The expression of IGF-2 and Arc mRNA and protein were respectively measured by using RT-PCR and Western blotting. RESULTS: There was an elevation in the plasma corticosterone level of rat model of maternal chronic stress during pregnancy. Maternal stress's offspring exposed to an enriched environment could decrease their plasma corticosterone level and improve their weight. The offspring of maternal stress during pregnancy exhibited abnormalities in Morris water maze and Y-maze, which were improved in an enriched environment. The expression of IGF-2, Arc mRNA, and protein in offspring of maternal stress during pregnancy was boosted and some relationships existed between these parameters after being exposed enriched environment. CONCLUSIONS The learning and memory impairment in offspring of prenatal stress can be rectified by the enriched environment, the mechanism of which is related to the decreasing plasma corticosterone and increasing hippocampal IGF-2 and Arc of offspring rats following maternal chronic stress during pregnancy.
Animals ; Cytoskeletal Proteins/metabolism* ; Female ; Gene Expression Regulation ; Hippocampus/metabolism* ; Insulin-Like Growth Factor II/metabolism* ; Learning ; Learning Disabilities/psychology* ; Male ; Memory Disorders/psychology* ; Nerve Tissue Proteins/metabolism* ; Pregnancy ; Prenatal Exposure Delayed Effects/psychology* ; Random Allocation ; Rats ; Rats, Wistar ; Social Environment ; Stress, Psychological/genetics*

Objective: To investigate the influence of high fat diet-induced obesity (HFDIO) on the differentially methylated region (DMR) of the imprinted gene and global genome methylation of sperm DNA. METHODS: We performed bisulfite sequencing on the DMR of the imprinted gene and global genome methylation of sperm DNA in the mouse model of HFDIO. RESULTS: No statistically significant differences were found between the HFDIO model and normal control mice in MEG3-IG (93.73 vs 97.26%, P = 0.252), H19 (98.00 vs 97.83%, P = 0.920), IGF2 (97.34 vs 96.25%, P =0.166), IGF2R (1.43 vs 1.11%, P = 0.695), PEG3 (0.19 vs 0.38%, P = 0.537), MEST (0.23 vs 0.68%, P = 0.315), NNAT (0.31 vs 0.00%, P = 0.134), or SNRPN (1.88 vs 3.13%, P = 0.628). A total of 8 942 DMRs were detected across the sperm genome (P <0.05). Gene functional enrichment analysis indicated that the enriched terms with the largest numbers of genes were the metabolic process (n = 1 482), RNA synthesis (n = 779), and transcription (n = 767). CONCLUSIONS The methylation level underwent no significant change in the DMRs of the imprinted genes from the mice with HFDIO, but the CG methylation of the genes involved in the metabolic process, RNA synthesis and transcription were significantly altered.
Animals ; DNA Methylation ; Diet, High-Fat ; Genome ; Genomic Imprinting ; Insulin-Like Growth Factor II ; Male ; Mice ; Obesity ; genetics ; metabolism ; RNA ; biosynthesis ; Spermatozoa ; metabolism

OBJECTIVETo explore the involvement of hepatitis B X protein (HBx) in promoter 3 (P3)-driven mRNA overexpression of the insulin-like growth factor II gene (IGF-II) and investigate the underlying epigenetic mechanism.

METHODSLevels of P3 and HBx mRNA and status of P3 methylation were analyzed in human hepatocellular carcinoma (HCC) samples, with and without hepatitis B virus (HBV) infection, using quantitative reverse transcription-PCR and bisulfite sequencing. In addition, the levels of P3 mRNA and P3 methylation were examined in HepG2 cells stably overexpressing HBx (HepG2-HBx). Finally, P3 promoter-luciferase constructs were cotransfected into HepG2 cells along with an HBx-expressing plasmid, and the effects of HBx on transcriptional activity and methylation of P3 were analyzed. Statistical analyses of the data were conducted by chi square test, Fisher's exact test, Student's t-test, Marn-Whitney U test, and Pearson's correlation coefficient test.

RESULTSThe HBV-positive HCC specimens had significantly higher levels of P3 mRNA than the HBV-negative HCC specimens (-9.59 ± 3.22 vs. -12.97 ± 3.08 delta CT; P=0.006) but significantly lower levels of P3 methylation (mean values for the 17 CpG sites (36.9% ± 15.5% vs. 52.1% ± 19.1%; P=0.025). The P3 transcript abundance was positively correlated with the level of HBx expression and negatively correlated with the level of P3 methylation. The epigenetic results from experiments with the HepG2-HBx cells were similar. Transfection of HBx significantly decreased P3 methylation level and increased its activity.

CONCLUSIONHBx expression may promote IGF-II expression by inducing hypomethylation of its P3 promoter in hepatocellular carcinoma.

Carcinoma, Hepatocellular ; genetics ; metabolism ; DNA Methylation ; Epigenesis, Genetic ; Female ; Gene Expression ; Hep G2 Cells ; Humans ; Insulin-Like Growth Factor II ; genetics ; metabolism ; Liver Neoplasms ; genetics ; metabolism ; Male ; Promoter Regions, Genetic ; RNA, Messenger ; genetics ; Trans-Activators ; pharmacology

BACKGROUNDThe insulin-like growth factor signaling pathway plays an important role in the modulation of cell growth and proliferation. The aim of this study was to investigate the role of polymorphisms of the insulin-like growth factor 2 (IGF2) and IGF-binding protein 3 (IGFBP3) genes, which encode key proteins of this pathway, as risk factors for gastric carcinoma (GC).

METHODSA case-control study including 404 histologically confirmed GC patients and 424 healthy controls of the same ethnicity was conducted to retrospectively investigate the genetic polymorphisms of two genes, IGF2+820A>G (rs680) and IGFBP3 A-202C (rs2854744). Adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using Logistic regression.

RESULTSThe IGF2 genetic variants examined contributed to GC risk individually (OR, 1.26; 95% CI, 1.08-1.46). The genotype frequencies of IGFBP3 A-202C were not significantly different between the cancer cases and controls (P > 0.05). Compared to the IGF2 AA genotype, carriers of one variant combined genotype were more pronounced among young subjects (<60 years), male subjects, never smokers, and those with a family history of cancer (OR = 1.36, 95% CI = 1.09-1.72, P < 0.05; OR = 1.61, 95% CI = 1.28-2.08, P < 0.05; OR = 1.46, 95% CI = 1.11-1.98, P < 0.05; OR = 1.53, 95% CI = 0.91-2.6, P < 0.05; respectively). Moreover, when the combined effects of the risk genotypes were investigated, significant associations were detected between highrisk genotypes in IGF2 and IGFBP3 (OR, 2.47; 95% CI, 1.75-3.49).

CONCLUSIONSOur results suggest that polymorphic variants of the IGF2 genes modulate gastric carcinogenesis. Moreover, when the IGF2 and IGFBP3 variants are evaluated together, a greater effect on GC risk is observed.

Adult ; Aged ; Case-Control Studies ; China ; Female ; Genetic Predisposition to Disease ; genetics ; Genotype ; Humans ; Insulin-Like Growth Factor Binding Protein 3 ; genetics ; Insulin-Like Growth Factor II ; genetics ; Logistic Models ; Male ; Middle Aged ; Polymorphism, Genetic ; genetics ; Stomach Neoplasms ; genetics

Insufficient epigenetic reprogramming of donor nuclei is believed to be one of the most important causes of low development efficiency of mammalian somatic cell nuclear transfer (SCNT). Previous studies have shown that both the in vitro and in vivo development of mouse SCNT embryos could be increased significantly by treatment with various histone deacetylase inhibitors (HDACi), including Trichostatin A, Scriptaid, and m-carboxycinnamic acid bishydroxamide (CBHA), in which only the effect of CBHA has not yet been tested in other species. In this paper we examine the effect of CBHA treatment on the development of porcine SCNT embryos. We have discovered the optimum dosage and time for CBHA treatment: incubating SCNT embryos with 2 μmol/L CBHA for 24 h after activation could increase the blastocyst rate from 12.7% to 26.5%. Immunofluorescence results showed that the level of acetylation at histone 3 lysine 9 (AcH3K9), acetylation at histone 3 lysine 18 (AcH3K18), and acetylation at histone 4 lysine 16 (AcH4K16) was raised after CBHA treatment. Meanwhile, CBHA treatment improved the expression of development relating genes such as pou5f1, cdx2, and the imprinted genes like igf2. Despite these promising in vitro results and histone reprogramming, the full term development was not significantly increased after treatment. In conclusion, CBHA improves the in vitro development of pig SCNT embryos, increases the global histone acetylation and corrects the expression of some developmentally important genes at early stages. As in mouse SCNT, we have shown that nuclear epigenetic reprogramming in pig early SCNT embryos can be modified by CBHA treatment.
Acetylation ; Animals ; Blastocyst ; cytology ; Cell Nucleus ; metabolism ; Cinnamates ; pharmacology ; Embryo, Mammalian ; drug effects ; metabolism ; Embryonic Development ; drug effects ; Epigenesis, Genetic ; Female ; Gene Expression ; Histone Deacetylase Inhibitors ; pharmacology ; Histones ; metabolism ; Homeodomain Proteins ; genetics ; metabolism ; In Vitro Techniques ; Insulin-Like Growth Factor II ; genetics ; metabolism ; Nuclear Transfer Techniques ; Octamer Transcription Factor-3 ; genetics ; metabolism ; Swine

BACKGROUNDPatients with single station mediastinal lymph node (N2) non-small cell lung cancer (NSCLC) have a better prognosis than those with multilevel N2. The molecular factors which are involved in disease progression remain largely unknown. The purpose of this study was to investigate gene expression differences between single station and multilevel N2 NSCLC and to identify the crucial molecular factors which are associated with progress and prognosis of stage N2 NSCLC.

METHODSGene expression analysis was performed using Agilent 4×44K Whole Human Genome Oligo Microarray on 10 freshfrozen lymph node tissue samples from single station N2 and paired multilevel N2 NSCLC patients. Real-time reverse transcription (RT)-PCR was used to validate the differential expression of 14 genes selected by cDNA microarray of which four were confirmed. Immunohistochemical staining for these validated genes was performed on formalin-fixed, paraffinembedded tissue samples from 130 cases of stage N2 NSCLC arranged in a high-density tissue microarray.

RESULTSWe identified a 14 gene expression signature by comparative analysis of gene expression. Expression of these genes strongly differed between single station and multilevel N2 NSCLC. Four genes (ADAM28, MUC4, CLDN1, and IGF2) correlated with the results of microarray and real-time RT-PCR analysis for the gene-expression data in samples from 56 NSCLC patients. Immunohistochemical staining for these genes in samples from 130 cases of stage N2 NSCLC demonstrated the expression of IGF2 and CLDN1 was negatively correlated with overall survival of stage N2 NSCLC.

CONCLUSIONSOur results suggest that the expression of CLDN1 and IGF2 indicate a poor prognosis in stage N2 NSCLC. Further, CLDN1 and IGF2 may provide potential targeting opportunities in future therapies.

Adult ; Aged ; Carcinoma, Non-Small-Cell Lung ; metabolism ; mortality ; pathology ; Claudin-1 ; analysis ; genetics ; Female ; Humans ; Immunohistochemistry ; Insulin-Like Growth Factor II ; analysis ; genetics ; Lung Neoplasms ; metabolism ; mortality ; pathology ; Male ; Middle Aged ; Neoplasm Staging ; Prognosis

To investigate whether epigenetic alterations in the insulin-like growth factor-II (IGF-II) gene that cause differential transcription or expression are correlated with onset and severity of hepatocellular carcinoma (HCC). Patient-matched specimens of HCC, paracancerous, and non-cancerous tissues were collected from 40 primary liver cancer patients. Epigenetic alterations in the promoter (P3) sequence of the IGF-II gene were analyzed by methylation-specific PCR (MSP) and IGF-II transcription was measured by RT-PCR. IGF-II protein expression and clinicopathological features were assessed by immunohistochemistry and microscopic observation. The rate of IGF-II P3 methylation was significantly lower in HCC tissues (0%) than in paracancerous tissues (vs. 47.5%; x2 = 24.918, P less than 0.001) and non-cancerous tissues (vs. 100%; x2 = 80.000, P less than 0.001). IGF-II mRNA expression was significantly higher in HCC tissues (100%) than in paracancerous tissues (vs. 52.5%; x2 = 24.918, P less than 0.001) and non-cancerous tissues (vs. 0%; x2 = 80.000, P less than 0.001). IGF-II protein expression was significantly higher in HCC tissues (82.5%) than in paracancerous tissues (vs. 45.0%; x2 = 12.170, P less than 0.001) and non-cancerous tissues (vs. 0%; x2 = 56.170, P less than 0.001). IGF-II overexpression in HCC was significantly associated with degree of differentiation, extent of infiltrated serosa, size of tumor, and HBV-positive infection status. Epigenetic alterations in the IGF-II gene regulate its transcription and expression and are closely associated with HCC development and progression.
Adult ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; CpG Islands ; genetics ; DNA Methylation ; Epigenesis, Genetic ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; Insulin-Like Growth Factor II ; genetics ; metabolism ; Liver ; metabolism ; pathology ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Middle Aged ; Polymerase Chain Reaction ; methods ; Promoter Regions, Genetic ; RNA, Messenger ; genetics ; metabolism ; Transcription, Genetic

OBJECTIVETo explore the feasibility of IGF2 imprinting system in target gene therapy for tumors.

METHODSThe mouse H19 enhancer, DMD and promoter H19 were amplified by PCR from mouse genomic DNA and then cloned into the plasmid pDC312. The EGFP and DT-A fragments were amplified by PCR and cloned into the recombinant plasmid, and then the shuttle plasmid were transfected into HEK293 cells together with the adenoviral vector Ad5, namely, Ad-EGFP and Ad-DT-A. Adenovirus hexon gene expression was applied to confirm the presence of adenovirus infections. The effect of the IGF2 imprinting system was tested by fluorescence microscopy. RT-PCR and Western blotting after transfection of the recombinant adenoviral vectors into cancer cells were used to show loss of IGF2 imprinting (LOI) and maintenance of IGF2 imprinting (MOI), respectively. The anti-tumor effect was assessed by MTT and flow cytometry after the HCT-8 (LOI). Human breast cancer cell line MCF-7 (MOI) and human normal gastric epithelial GES-1 (MOI) cell line were transfected with Ad-DT-A in vitro. The anti-tumor effect was detected by injecting the Ad-DT-A in nude mice carrying HCT-8 tumors.

RESULTSThe expression of EGFP protein, DT-A mRNA and DT-A protein were seen to be positive only in the HCT-8 tumor cell line. Infection with Ad-DT-A resulted in obviously growth inhibition in HCT-8 cells (75.4 ± 6.4)% compared with that in the control group, and increased the percentage of apoptosis in the HCT-8 cells (20.8 ± 5.9)%. The anti-tumor effect was further confirmed by injecting the recombinant adenoviruses in HCT-8 tumor-bearing nude mice, and the results showed that the Ad-DT-A inhibited the tumor growth, with on inhibition rate of 36.4%.

CONCLUSIONSThe recombinant adenoviruses carrying IGF2 imprinting system and DT-A gene have been successfully constructed, while Ad-DT-A can effectively kill the tumor cells showing loss of IGF2 imprinting. It might play an important role in future target gene therapy against malignant tumors based on loss of IGF2 imprinting.

Adenoviridae ; genetics ; Animals ; Apoptosis ; Breast Neoplasms ; genetics ; pathology ; Colonic Neoplasms ; genetics ; pathology ; therapy ; Diphtheria Toxin ; biosynthesis ; genetics ; Female ; Genetic Therapy ; methods ; Genetic Vectors ; Genomic Imprinting ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Humans ; Insulin-Like Growth Factor II ; genetics ; metabolism ; MCF-7 Cells ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Peptide Fragments ; biosynthesis ; genetics ; Plasmids ; RNA, Messenger ; metabolism ; Random Allocation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection

OBJECTIVETo investigate the selective killing effect of herpes simplex virus-thymidine kinase/ganciclovir (HSV-tk/GCV) suicide gene system controlled by human IGF-II P4 promoter on HCC cells in vitro.

METHODSRecombinant shuttle plasmid vectors driven by IGF-II P4 promoter and driven by CMV promoter were constructed by techniques of gene recombination. The recombinant shuttle plasmids were then transfected into HepG2 and HeLa cells by techniques of lipidosome transfection. EGFP expression was detected by fluoroscopy. Tk and EGFP mRNA expression were detected by RT-PCR. The selective killing effect after GCV application was determined with MTT method. Statistical analysis was performed with ANOVA analysis.

RESULTSIdentification of pDC316-tkEGFP-P4 by enzyme digestion and sequencing analysis showed that the construction of the recombinant shuttle plasmid was correct. It was found that green fluorescence protein could only be seen in HepG2 cells, not in HeLa cells. The results of RT-PCR showed only two bands could be seen in the samples of pDC316-tkEGFP-P4 transfected HepG2 cells. The growth of HepG2 cells transfected with pDC316-tkEGFP-CMV and pDC316-tkEGFP-P4 were inhibited remarkably, the growth inhibition rates were 6.95% +/- 0.67%, 24.99% +/- 1.53%, 49.68% +/- 1.68%, 71.85% +/- 3.28% and 4.83% +/- 0.35% vs 17.34% +/- 1.15%, 30.17% +/- 1.30%, 40.39% +/- 0.82% (F = 24.055, P < 0.05), respectively. The growth of HeLa cells transfected with pDC316-tkEGFP-CMV were also inhibited, the growth inhibition rates were 6.36% +/- 0.83%, 23.95% +/- 1.72%, 45.13% +/- 1.64% and 69.38% +/- 3.17%, respectively. The growth of HeLa cells transfected with pDC316-tkEGFP-P4 was not inhibited. The growth inhibition rates were 0.91 +/- 0.04, 1.18 +/- 1.32, 1.19 +/- 0.10 and 1.32 +/- 0.05 (F = 26.469, P < 0.01) , respectively.

CONCLUSIONThe shuttle plasmid vector carrying the tkEGFP fusion protein gene driven by IGF-II P4 promoter has been constructed successfully and its specific expression in HepG2 cells provided the basis for targeted gene therapy for HCC.

Cell Line, Tumor ; Genes, Transgenic, Suicide ; genetics ; Genetic Vectors ; Humans ; Insulin-Like Growth Factor II ; genetics ; pharmacology ; Plasmids ; Promoter Regions, Genetic ; Thymidine Kinase ; genetics ; Transfection

The study investigated the effects of adenovirus-mediated gene transfection of basic fibroblast growth factor (bFGF), bFGF combined with interleukin-1 receptor antagonist protein (IL-Ra) and/or insulin-like growth factor-1 (IGF-1) both in human osteoarthritis (OA) chondrocytes and rabbits OA model. Human OA chondrocytes were delivered by adenovirus-mediated bFGF, IL-Ra and IGF-1 vectors, respectively. Chondrocyte proliferation, glycosaminoglycan (GAG) content, expression of type II collagen, ADAMTS-5, MMP-13, MMP-3 and TIMP-1 were determined. Rabbit OA model was induced by anterior cruciate ligament transaction (ACLT) in knees. Adenoviral vectors encoding human bFGF, IL-Ra and IGF-1 were injected intraarticularly into the knee joints after ACLT. The effects of adenovirus- mediated gene transfection on rabbit OA were evaluated. In vitro, the transfected genes were expressed in cell supernatant of human OA chondrocytes. AdbFGF group significantly promoted chondrocyte proliferation, and increased GAG and type II collagen synthesis than in the OA group. As two or three genes were transfected in different combinations, there was significant enhancement on the GAG content, type II collagen synthesis, and TIMP-1 levels, while ADAMTS-5, MMP-13, and MMP-3 levels were reduced. In vivo, the transfected genes were expressed in synovial fluid of rabbits. Intraarticular delivery of bFGF enhanced the expression of type II collagen in cartilage and decreased cartilage Mankin score compared with the OA control group (P = 0.047; P < 0.01, respectively). Multiple-gene transfection in different combinations showed better results than bFGF transfection alone. This study suggests that bFGF gene transfection is effective in treating experimental OA. Multiple gene transfection has better biologic effects on OA.
Adenoviridae/*genetics ; Animals ; Chondrocytes/drug effects/*metabolism ; Collagen Type II/genetics/metabolism ; Fibroblast Growth Factor 2/*genetics ; Gene Therapy/methods ; Genetic Vectors/administration & dosage/*genetics ; Humans ; Insulin-Like Growth Factor I/*genetics/metabolism ; Interleukin 1 Receptor Antagonist Protein/*genetics/metabolism ; Interleukin-1/genetics/metabolism ; Matrix Metalloproteinase 13/genetics/metabolism ; Matrix Metalloproteinase 3/genetics/metabolism ; Osteoarthritis/*therapy ; Rabbits ; Tissue Inhibitor of Metalloproteinase-1/genetics ; Transfection