OBJECTIVEIntrauterine growth retardation (IUGR) may contribute to the disorder of development of fetal brains. L-arginine has been known to be effective in blood vessel distension and improving the blood circulation of placentas. Recent studies have shown that L-arginine can ameliorate the placental hypoxia and improve the development of fetus. This study aimed to explore the effects of L-arginine on the expression of insulin-like growth factor (IGF)-I, IGF-II, IGF binding protein-3(IGFBP3)and IGF-I mRNA in brains of IUGR rats and the possible mechanisms of L-arginine.

METHODSThirty-six pregnant rats were randomly assigned into four groups: Control, Model, Low dose L-arginine (100 mg/kg) and High-dose L-arginine (200 mg/kg L-arginine) groups (n=9 each). IUGR was induced by passive smoking in rats from the last three groups. L-arginine was administered for the last two groups between days 8 and 20 of gestation. On day 21 of gestation, the pup rats were delivered by cesarean section. The levels of IGF-I, IGF-II and IGFBP3 in the brains of pup rats were measured by enzyme-linked immunoadsordent assay (ELISA) and the expression of IGF-I mRNA was detected by fluorescence quantitative PCR (FQ-PCR).

RESULTSThe levels of IGF-I, IGF-II and IGF-I mRNA expression in the Model group were significantly lower than in the Control group, with the IGF-I levels of 0.789 +/- 0.062 ng/mg vs 0.947 +/- 0.042 ng/mg, the IGF-II levels of 0.270 +/- 0.020 ng/mg vs 0.374 +/- 0.015 ng/mg and the IGF-I mRNA expression of (13.12 +/- 1.39) x 10(4) cps/mug RNA vs (21.28 +/- 3.54) x 10(4) cps/mug RNA (P < 0.01). In contrast, the IGFBP3 levels in the Model group were significantly higher than in the Control group (0.253 +/- 0.011 ng/mg vs 0.089 +/- 0.015 ng/mg; P < 0.01). Low or high dose L-arginine treatment increased significantly the IGF-I levels from 0.789 +/- 0.062 ng/mg (Model group) to 0.937 +/- 0.067 ng/mg (low dose group) or 0.858 +/- 0.077 ng/mg (high dose group), the IGF-II levels from 0.270 +/- 0.020 ng/mg (Model group) to 0.318 +/- 0.018 ng/mg (low dose group) or 0.354 +/- 0.021 ng/mg (high dose group) and the IGF-I mRNA expression from (13.12 +/- 1.39) x 10(4) cps/mug RNA (Model group) to (19.24 +/- 2.48) x 10(4) cps/mug RNA (low dose group) or (17.35 +/- 2.30) x 10(4) cps/mug RNA (high dose group) (P < 0.01). The IGFBP3 levels were significantly reduced after low or high dose L-arginine treatment (0.132 +/- 0.006 ng/mg or 0.146 +/- 0.009 ng/mg) compared with those of the Model group (0.253 +/- 0.011 ng/mg) ( P < 0.01).

CONCLUSIONSL-arginine can increase the levels of IGF-I and IGF-II and the IGF-I mRNA expression, and decrease the IGFBP3 level in the brain of rats with IUGR induced by passive smoking, thereby offering protective effects against IUGR.

Animals ; Arginine ; pharmacology ; therapeutic use ; Female ; Fetal Growth Retardation ; metabolism ; prevention & control ; Insulin-Like Growth Factor Binding Protein 3 ; analysis ; Insulin-Like Growth Factor I ; analysis ; genetics ; Insulin-Like Growth Factor II ; analysis ; Male ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar

To explore the relationship between Insulin-like growth factor (IGF)-I , -II and lung development in neonatal rats. 80 timed pregnant Sprague-Dawley (SD) rats were randomly divided into 4 groups (n=20): group A (Control group), group B (Dexamethasone (DEX) 1 group), group C (DEX 2 group), group D (retinoic acid (RA) group). 20 pregnant rats in group A, B and D were injected subcutaneously or intraperitoneally with vehicle (NS), DEX, or RA respectively during gestational day 16 to 18. All newborn rats in group C were subcutaneously injected with DEX at day 1 to 3 after birth. The lung tissue was obtained at the following times: fetuses at gestational ages of 18, 20 and 21 days, and 1, 3, 5, 7, 10, 14 and 21 days after birth. Lung tissues were used for histopathological study, the polypeptides analysis of IGF- I, -II (immunohistochemistry and Western blot) and mRNA analysis ( RT- PCR). The results showed that the strongest expression of IGF- I in group A and D occurred at ages of 5-7 days (alveolar stage). The stronger their expressions, the better the alveolar develop. The peak stage of expression in group B occurred earlier, on the day 3 after birth. Compared with group A, the expression of IGF-I during gestation age of 18 days to age of 3 days in group B were significantly higher (P<0.01), but significantly lower at other time points (P<0.01). The expression of IGF-I was lower in group C all the time and always higher in group D than those in group A (P<0.01). The peak expression of IGF-II took place at the gestation age of 18 days, then gradually dropped to trace. During 18 days of gestation to age of 3 days, the expression of IGF-II in group B was significantly higher than that in group A (P<0.01). No difference was found among all other groups. The change in the expression of IGF-I, -II mRNA in all 4 groups was similar to that of their polypeptides. The results suggested that there is a close linking between IGF-I , -II and lung development in newborns. The IGF-II works at early stage and the that of IGF- I works at the stage of new septa formation and alveoli maturation. The stronger their expressions, the more mature the lung development.
Animals ; Animals, Newborn ; Dexamethasone ; pharmacology ; Female ; Insulin-Like Growth Factor I ; biosynthesis ; genetics ; Insulin-Like Growth Factor II ; biosynthesis ; genetics ; Lung ; embryology ; growth & development ; metabolism ; Male ; Pregnancy ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Tretinoin ; pharmacology

Animals ; Cells, Cultured ; Dioxins ; pharmacology ; Insulin-Like Growth Factor II ; genetics ; metabolism ; Osteoblasts ; drug effects ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar

OBJECTIVETo study the imprinting status of IGF2 and phenotypes of loss of imprinting (LOI) in cord blood of neonates of Chinese Han population and to investigate relative factors to LOI.

METHODSCord blood of 1010 Chinese Han newborns were collected and the imprinting status of IGF2 was detected by reverse transcription-PCR(RT-PCR) and restriction fragment length polymorphism.The relationships between LOI and fetal growth indices, features of parents and grandparents, clinical characteristics were analyzed.

RESULTSOf all cases, 42.8% (432/1010) were heterozygous for a polymorphism of Apa I site in exon 9 of IGF2, while 21.6%(66/306) displayed IGF2 LOI. Maternal factors including average age, gestational age, BMI pre-pregnancy, weight gain during pregnancy and the level of HB, HCT, and other indices of biochemistry in their second and third trimester were not correlated with LOI expression. However in newborns with fathers older than 35 yrs, 31.7%(19/60) displayed LOI, which was significantly more common than that in newborns with younger fathers (P< 0.05, chi square is 4.69). There were no difference in birth weight (BW) between normal imprinting and LOI groups. But if the newborn's weights were in 2500-2999 g, LOI was 6.25%(2/32), which was significantly lower than that in 3000 g group (P< 0.05, chi square is 4.89). In groups with BW being less than 2500 g and more than/equal to 4000 g, the LOI newborn's blood glucose was decreased significantly after 2 hrs (P< 0.01, t is 7.47 and 10.9).

CONCLUSIONIn newborns of Chinese Han population, 21.6% showed IGF2 LOI in cord blood. IGF2 LOI may have some influences on fetal growth. Paternal age is associated with LOI.

Asian Continental Ancestry Group ; genetics ; China ; Female ; Fetal Blood ; metabolism ; Genomic Imprinting ; genetics ; Humans ; Infant, Newborn ; Insulin-Like Growth Factor II ; genetics ; Pregnancy ; Reverse Transcriptase Polymerase Chain Reaction

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 study the imprinting status and expression level of insulin-like growth factor 2 (IGF2) gene in colorectal cancer and to provide a clue for the mechanism of carcinogenesis of colorectal cancer.

METHODSThe expression levels of IGF2 in the paired colorectal cancer and adjacent normal tissue were examined and compared by use of semi-quantitative reverse transcription-polymerase chain reaction. The imprinting status of IGF2 was detected by restriction fragment length polymorphism. The relationships between the expression level of IGF2, its imprinting status, and the carcinogenesis of colorectal cancer were analyzed.

RESULTSIGF2 was overexpressed in 82.4% (28/34) of colorectal cancer tissues which was significantly higher than those of the matched normal tissues (P<0.01, t=3.01). 87.5% (14/16) of colorectal cancer showed loss of imprinting(LOI), while 71.4%(10/14) of normal tissues also displayed LOI of IGF2.

CONCLUSIONOverexpression of IGF2 was found to play an important role in carcinogenesis of colorectal cancer. LOI of IGF2 may be a prophase manifestation of colorectal cancer.

Colorectal Neoplasms ; genetics ; pathology ; DNA, Neoplasm ; genetics ; metabolism ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Genomic Imprinting ; Humans ; Insulin-Like Growth Factor II ; genetics ; Male ; Middle Aged ; RNA, Neoplasm ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo study the effects of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) on the development of fetal rats and to explore the relationship between TCDD-induced abnormal development in rats and the expression and the methylation of insulin-like growth factor 2 gene (Igf2).

METHODSA single dose of 10 microg/kg TCDD was given to gestation day (GD) 10 pregnant rats by gavage. On GD20, the fetuses were taken out and examined. The crown-rump length, the body weight and the placental weight were measured. The expression of Igf2 in liver was detected by real-time quantitative reverse transcription (RT-PCR) and Western blot. The methylation of Igf2 differentially methylated regions (DMRs) in liver was analyzed by a methylation-sensitive restriction enzyme Hpa II PCR assay and a bisulfite-modified DNA sequencing procedure.

RESULTSIn the treatment group, 12.2% of the fetuses were either dead or absorbed, and 11.6% of them were malformed. For the live fetuses, their crown-rump length, body weight and placental weight were significantly lower than those of the control group. The relative amount of Igf2 mRNA in the treated livers and the control livers was 0.77 +/- 0.11 and 0.27+/- 0.15, respectively. The number was significantly higher in the treatment group than in the control group (P < 0.01). Western blot also showed a remarkable up regulation of Igf2 protein in liver after treatment. The two groups showed no difference in the methylation status of Igf2 DMR1 in liver. The DMR2 Igf2 was significantly hypomethylated in the treated livers than in the control livers.

CONCLUSIONExposure to TCDD in pregnancy can lead fetal rats to death, absorption, malformation and intrauterine growth retardation (IUGR). The TCDD led abnormal development in rats may be associated with the hypomethylated DMR2 of Igf2 and the up regulation of Igf2 in liver.

Animals ; Blotting, Western ; DNA Methylation ; Female ; Fetal Growth Retardation ; chemically induced ; genetics ; metabolism ; Gene Expression ; drug effects ; Insulin-Like Growth Factor II ; genetics ; metabolism ; Polychlorinated Dibenzodioxins ; toxicity ; Pregnancy ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction

PTEN/MMAC1 is a tumor suppressor gene that is mutated in a variety of advanced and metastatic cancers. Its major function is likely to be the phosphatase activity that regulates the phosphotidylinositol (PI)3-kinase/ Akt pathway. On the other hand, IGF system plays an important role in cell proliferation and cell survival via PI3-kinase/Akt and mitogen-activated protein kinase pathways in many cancer cells. To evaluate effect of PTEN on cell growth and IGF system in gastric cancer, human gastric adenocarcinoma cells (SNU-5 & -216) were transfected with human PTEN cDNA. Those PTEN- transfected gastric cancer cells had a lower proliferation rate than the pcDNA3-transfected cells. PTEN overexpression induced a profound decrease in the IGF-II and IGF-IR expression levels, and downregulation of IGF-II expression by PTEN was mediated through the regulation of the IGF-II promoter. In addition, a PI3-kinase inhibitor, LY294002, induced the downregulation of IGF-II expression. The PTEN-overexpressing SUN-5 and -216 cells were more sensitive to death induced by etoposide and adriamycin that induce DNA damage than the pcDNA3-transfected cells. These findings suggest that PTEN suppresses the cell growth through modulation of IGF system and sensitizing cancer cells to cell death by anticancer drugs.
Antineoplastic Agents/*pharmacology ; Cell Line, Tumor ; Cell Proliferation/drug effects ; *Down-Regulation ; Humans ; Insulin-Like Growth Factor II/*genetics/*metabolism ; PTEN Phosphohydrolase/genetics/*metabolism ; Receptor, IGF Type 1/genetics/metabolism ; Research Support, Non-U.S. Gov't ; Stomach Neoplasms/*genetics/metabolism/*pathology

OBJECTIVETo study the effect of angiotensin II (Ang II) on pregnancy-associated plasma protein A (PAPP-A) and insulin-like growth factor 1 (IGF-1) mRNA expressions in human umbilical artery smooth muscle cells (HUVSMCs).

METHODSIn the presence or absence of Ox-LDL, HUVSMCs were cultured with Ang II of 10(-5) mol/L for 0, 6, 12, 24, 36, and 48 h, or with Ang II at the concentrations of 10(-7), 10(-6), 10(-5), and 10(-4) mol/L for 24 h, after which the cells were then collected to detect PAPP-A and IGF-1 mRNA expressions in the cells using RT-PCR.

RESULTSAt the concentration of 10(-5) mol/L, Ang II showed a time-dependent effect in inducing PAPP-A and IGF-1 mRNA expressions, which began to increase at 12 h of culture and reaching the highest level at 24 h. Ang II also dose-dependently induced PAPP-A and IGF-1 mRNA expressions, and 10(-5) mol/L Ang II induced the highest expression levels of the two genes. Ox-LDL exposure significantly further increased the expression levels of PAPP-A and IGF-1 mRNA in the cells regardless of the Ang II concentration or duration for cell treatment (P<0.05).

CONCLUSIONAng II can time- and dose-dependently induces PAPP-A and IGF-1 mRNA expression in HUVSMCs and is responsible for inducing platelet activity and inflammatory reaction in acute coronary syndromes, and the effects of Ang II can be enhanced by Ox-LDL.

Angiotensin II ; pharmacology ; Cells, Cultured ; Drug Synergism ; Humans ; Insulin-Like Growth Factor I ; genetics ; metabolism ; Lipoproteins, LDL ; pharmacology ; Myocytes, Smooth Muscle ; cytology ; metabolism ; Pregnancy-Associated Plasma Protein-A ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Umbilical Arteries ; cytology ; 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