Expression Of H19 Gene Research Articles

Investigating the effect of Quercetin in the presence of CoCl2 as an inducing hypoxia agent on the biological characteristics of human telomerase reverse transcription-immortalized adipose tissue-derived MSCs

Studying the effect of small chemical molecules on stem cell characteristics under normoxia and hypoxia conditions is crucial to discovering the best conditions for effective biomedical applications. This study aimed to investigate the effect of Quercetin (QC; a flavonoid) in the presence of CoCl2 as a mimicking hypoxia chemical on the biological features of human telomerase reverse transcription-immortalized mesenchymal stem cell (hTERT-MSC) lines. The effect of CoCl2, QC, and their combination on the viability, proliferation, and migration of hTERT-MSCs were evaluated by MTT, Trypan-blue staining and cell counting by hemocytometer, and in vitro wound healing assays, respectively. Moreover, the effect of treatments on the reactive oxygen species (ROS) production, cell cycle, and HIF1a, c-MET, H19, and CASP3 gene expression was assessed by NBT, PI-staining and flow-cytometry, and real-time PCR assays, respectively. We found that CoCl2 and QC have different effects on the viability, proliferation, and migration of hTERT-MSCs in a dose-dependent manner. In addition, CoCl2 and QC affect ROS levels in cells in a dose- and time-dependent manner. While CoCl2 up-regulated HIF1a, QC and CoCl2 down-regulated CASP3 and c-MET in hTERT-MSCs. Moreover, QC reduced HIF1a and lncRNA-H19 expression in cells. Furthermore, in the presence of CoCl2, QC at low concentrations reduced hTERT-MSC survival, proliferation, and migration at 48 h; however, at high concentrations, it induced cell survival and proliferation. The combination treatment also up-regulated ROS levels and down-regulated the investigated genes in cells. Altogether, we conclude that QC at high concentrations under CoCl2-mediated hypoxia and short exposure time induces hTERT-MSCs survival and proliferation.

Effects of TABATA Exercise Volume and Royal Jelly Supplementation on NLRP3 Inflammasome and lncRNA-H19 Expression in Obese Men

Background: Both coding and long non-coding RNAs (lncRNAs) have emerged as vital regulators in almost every cellular process, and their expression can be modulated by external stimuli, such as physical exercise. Objectives: The current research aimed to investigate the effects of different volumes of TABATA-high-intensity interval training (HIIT) exercises combined with royal jelly (RJ) supplementation on the NLRP3 inflammasome and lncRNA-H19 expression in obese males. Methods: Forty-two healthy men [Body Mass Index (BMI) = 30 kg/m², waist-to-hip ratio = 0.95, age range: 40 - 60 years] volunteered to participate in the study. The individuals were randomly divided into five experimental groups (N = 35) and one control + placebo group (N = 7). The high-volume (HV) or low-volume (LV) TABATA exercise programs were performed twice a week for 8 weeks. Participants in the RJ supplementation groups received a 1000 mg capsule once a day for 8 weeks. The expression of NLRP3 and lncRNA-H19 genes was evaluated using the real-time PCR method. Results: The NLRP3 gene expression in the Bruce test, measured before and after the 8-week exercise interventions and RJ supplementation, showed insignificant changes across the different groups. However, the H19 gene expression in the Bruce test showed a significant reduction in the HV-TABATA HIIT intervention groups, which was more pronounced than in the LV groups after 8 weeks: HV group (P = 0.004), RJ group (P = 0.001), HV + RJ group (P = 0.007), and LV + RJ group (P = 0.002). After 8 weeks of non-pharmacological interventions involving exercise training and supplementation, a significant decrease in NLRP3 and a significant increase in H19 gene expression were detected in the HV group compared to the LV group (P = 0.05 and P = 0.010). Significant improvement was also found in the resting H19 levels between the RJ and LV groups (P = 0.011) and the LV + RJ group (P = 0.44). Moreover, a significant reduction in resting NLRP3 gene expression was observed between the RJ + LV and LV groups (P = 0.038). Conclusions: Chronic HV TABATA HIIT exercise, when combined with RJ supplementation, is effective in attenuating inflammatory responses to acute stress.

The Expression of TP53TG1, LINC00342, MALAT1, H19, and MEG3 Long Noncoding RNAs in Type 2 diabetes mellitus

Type 2 diabetes is a complex and multifactorial metabolic disorder. The frequency of type 2 diabetes has dramatically increased worldwide. Long noncoding RNAs play a regulatory role in pathological processes of type 2 diabetes. The aim of the study was to analyze TP53TG1, LINC00342, MALAT1, H19, and MEG3 lncRNAs in patients with type 2 diabetes and metabolic parameters, as well as the risk of diabetic retinopathy. Participants included 51 patients with diabetes and 70 healthy individuals. The expression of the TP53TG1 and LINC00342 genes was significantly decreased in the patients with diabetes compared to healthy individuals. MALAT1 gene expression was higher in diabetes patients. H19 gene expression was increased in the patients with diabetic retinopathy compared patients without retinopathy. TP53TG1, LINC00342, and MEG3 expression was decreased in patients with diabetic retinopathy and MALAT1 expression was increased. H19 is positively correlated with triglyceride levels; TP53TG1 and LINC00342 are positively correlated with HbA1c levels and fasting glucose levels. MALAT1 is negatively correlated with HDL levels and positively correlated with LDL levels. A decrease in the expression level of TP53TG1 and LINC00342 and an increase in the level of MALAT1 in diabetes, as well as an association with glycemic control, indicate the role of the studied noncoding RNAs in the development of type 2 diabetes mellitus and retinopathy and can be considered as candidates for early diagnosis of type 2 diabetes.

Long Non-Coding RNA H19 Expression in Leukemia Patients

Abstract Objective: BCR-ABL fusion gene occurs with the formation of translocations in the t(9;22) region of the Philadelphia (Ph) chromosome, which is used as a diagnostic biomarker in Chronic Myeloid Leukemia (CML). These abnormal genetic changes, which cover 15% of leukemias, reach dimensions that threaten human life. Recent studies have determined that thousands of genes expressed in differentiation and development processes contain non-protein-coding RNA with a regulatory role. Of these, the first discovered long noncoding RNA (LncRNA) H19 has been associated with its biological role, cell proliferation, apoptosis and metastasis Therefore, studies have been conducted considering that LncRNA H19 can be used as a biological marker in CML patients. Method: For this study, blood from 72 CML patients over 18 years of age and 64 healthy individuals were used. After RNA isolation of each of these bloods and cDNAs were obtained, the expression levels of the LncRNA H19 gene were analyzed by Real Time PCR method. Results: As a result of the expression study, it was found that LncRNA H19 gene expression increased 4.37 times and was upregulated in Bcr-Abl positive patients (p=0.414683). Conclusion: In this study conducted in Diyarbakır region, we think that LncRNA H19, which is up-regulated in terms of CML profiles, can be used as a biological marker for new treatment applications.

Expression analysis of vitamin D receptor-associated long noncoding RNAs in patients with relapsing-remittingmultiple sclerosis.

Vitamin D is a neuroactive steroid that carries out its biological functions through the vitamin D receptor (VDR). The VDR gene interacts with certain long noncoding RNAs (lncRNAs). The present study is aimed at evaluating the expression levels of the VDR gene as well as those of HOTAIR, H19, MALAT1, and P21 lncRNAs in patients with relapsing-remitting multiple sclerosis (RRMS). This research was conducted on 38 RRMS patients and 38 healthy individuals. The expression levels of VDR and selected lncRNAs in peripheral blood as well as those of vitamin D in the plasma were measured. The results revealed a significant increase in the expression of lncRNA H19 in the RRMS group compared to the control group. The analysis of the receiver operating characteristic (ROC) curve for H19 gene expression demonstrated a diagnostic value of 0.699 (95% CI: 0.575-0.823). Positive correlations were detected between VDR and lncRNA HOTAIR (r = 0.446, p = 0.008), H19 (r = 0.351, p = 0.042), MALAT1 (r = 0.464, p = 0.006), and P21 (r = 0.512, p = 0.002) in MS patients. The findings of this study suggest that lncRNA H19 could serve as a potential biomarker for MS diagnosis (Tab. 4, Fig. 1, Ref. 34).

Association among urinary polycyclic aromatic hydrocarbons metabolites, SAHH activity and H19 expression in coke oven workers

Objective: To investigate the relationship of polycyclic aromatic hydrocarbons (PAHs) exposure, S-adenosylhomocysteine hydrolase (SAHH) activity and long noncoding RNA H19 gene expression in the urine of coke oven workers. Methods: In September 2019, in a coking plant in Taiyuan City, 146 male workers who had worked in coke oven operations for one year were selected through a completely random sampling method, and their basic personal information was collected by questionnaire survey, and blood and urine samples were collected. The levels of 4 PAHs metabolites 2-hydroxfluorene (2-FLU), 2- hydroxynaphthalene (2-NAP), 9-hydroxyphenanthren (9-PHE), and 1-hydroxypyrene (1-OHP) in urine were detected by high performance liquid chromatography (HPLC) -fluorescence detection method. HPLC-UV detection method was used to detect the content of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in plasma, and the SAHH activity value was obtained by calculating the ratio. Reverse transcription PCR method was used to determine the H19 gene expression level. Urine levels of 2-FLU, 2-NAP, 9-PHE, and 1-OHP were divided into Q(1), Q(2), Q(3), and Q(4) groups according to quartiles (P(25), P(50), P(75)). Regression, trend test and restricted cubic splines were used to analyze the relationship among PAHs metabolites, SAHH activity, H19 gene expression and their dose-response. Results: The median age of coke oven workers was 39.60 years old, the median length of service was 20.38 years, and the urinary levels of 2-FLU, 2-NAP, 9- PHE, and 1-OHP were 0.29, 0.74, 0.09, and 0.06 μg/mmol Cr, respectively. The levels of 2-FLU, 2-NAP and 9-PHE in the urine of workers were significantly different between groups with different 1-OHP levels (P<0.05). After adjusting for age, length of service, smoking, drinking, and levels of 2-FLU, 2-NAP and 9-PHE, SAHH activity decreased with the increase of urinary 1-OHP level (OR=0.63, 95%CI: 0.41-0.98, P=0.038), showing a nonlinear relationship (P(nonlinear)= 0.030). H19 gene expression increased with the increase of urinary 1- OHP level (OR=1.51, 95%CI: 1.03-2.19, P=0.033), there was a linear relationship (P(trend)= 0.058). The relationship between the other three metabolites in urine and SAHH activity and H19 gene expression was not statistically significant (P>0.05) . Conclusion: Urinary 1-OHP level may be a risk factor for decreased SAHH activity and increased H19 gene expression in coke oven workers.

Analysis of Long Non-Coding RNA (lncRNA) UCA1, MALAT1, TC0101441, and H19 Expression in Endometriosis.

Endometriosis is a disease of complex etiology. Hormonal, immunological, and environmental factors are involved in its formation. In recent years, special attention has been paid to genetic mechanisms that can have a significant impact on the increased incidence of endometriosis. The study aimed to analyze the expression of four long non-coding RNA (lncRNA) genes, UCA1, MALAT1, TC0101441, and H19, in the context of the risk of developing endometriosis. The material for genetic testing for the expression of lncRNA genes were tissue slices embedded in paraffin blocks from patients with endometriosis (n = 100) and the control group (n = 100). Gene expression was determined by the RT-PCR technique. The expression of the H19 gene in endometriosis patients was statistically significantly lower than in the control group. A statistically significant association was found between H19 gene expression in relation to The Revised American Society for Reproductive Medicine classification of endometriosis (rASRM) in the group of patients with endometriosis. Research suggests that H19 expression plays an important role in the pathogenesis of endometriosis.

Evaluation of H19, Mest, Meg3, and Peg3 genes affecting growth and metabolism in umbilical cord blood cells of infants born to mothers with gestational diabetes and healthy mothers in Rafsanjan City, Iran.

Hyperglycemia during the first trimester leads to an increased risk of innate malformations as well as death at times close to delivery dates. The methylated genes include those from paternal H19 and PEG3 and those from maternal MEST and MEG3 that are necessary for the growth and regulation of the human fetus and its placenta. The aim of this study was to evaluate and compare the expression of these genes in the cord blood of healthy infants born to mothers with gestational diabetes mellitus (GDM) and healthy mothers.This case-control study was conducted on the cord blood of 40 infants born to mothers with GDM and 35 infants born to healthy mothers. Mothers were identified by measuring oral glucose tolerance in the 24th-26th week of pregnancy. Cord blood was obtained post-delivery, and cord blood mononuclear cells were immediately extracted, using Ficoll solution. Then, RNA extraction and cDNA synthesis were performed, and gene expression of MEG3, PEG3, H19, and MEST was assessed through quantitative real-time PCR.Findings show that the expression levels of MEG3, PEG3, H19, and MEST genes were significantly decreased in mononuclear cord blood cells of infants born to mothers with GDM when compared to those of the healthy control group.These findings reveal that the reduction of imprinted genes in mothers with GDM is most likely due to changes in their methylation by an epigenetic process. Considering the importance of GDM due to its high prevalence and its side effects both for mother and fetus, recognizing their exact mechanisms is of high importance. This has to be studied more widely.

Orientation of mouse H19 ICR affects imprinted H19 gene expression through promoter methylation-dependent and -independent mechanisms

The mouse Igf2/H19 locus is regulated by genomic imprinting, in which the paternally methylated H19 imprinting control region (ICR) plays a critical role in mono-allelic expression of the genes in the locus. Although the maternal allele-specific insulator activity of the H19 ICR in regulating imprinted Igf2 expression has been well established, the detailed mechanism by which the H19 ICR controls mono-allelic H19 gene expression has not been fully elucidated. In this study, we evaluated the effect of H19 ICR orientation on imprinting regulation in mutant mice in which the H19 ICR sequence was inverted at the endogenous locus. When the inverted-ICR allele was paternally inherited, the methylation level of the H19 promoter was decreased and the H19 gene was derepressed, suggesting that methylation of the H19 promoter is essential for complete repression of H19 gene expression. Unexpectedly, when the inverted allele was maternally inherited, the expression level of the H19 gene was lower than that of the WT allele, even though the H19 promoter remained fully hypomethylated. These observations suggested that the polarity of the H19 ICR is involved in controlling imprinted H19 gene expression on each parental allele, dependent or independent on DNA methylation of the H19 promoter.

P–338 Epigenetic alterations in H19-DMR regulatory region in endometrial tissues of women with endometriosis

Abstract Study question Is epigenetic modifications pattern in DMR region of H19 gene different in endometrial tissues of women with endometriosis in compare to normal endometrium? Summary answer The methylation level in DMR region of H19 gene was significantly lower in the endometriosis group. What is known already Endometriosis is characterized by the growth of endometrial-like tissue outside the uterus and has been considered as an epigenetic disease. The lncRNA H19 and insulin-like growth factor–2 (IGF2) genes form a reciprocally imprinted cluster (IGF2/H19). The expression of these two genes is regulated by imprinting control region (ICR). The ICR region is located between these genes and is a differentially methylated region (DMR). The H19 and IGF2 genes are involved in regulating cellular growth and differentiation and might be targeted by MeCP2 (a marker of DNA methylation) for subsequent epigenetic modifications through DMR regulatory region. Study design, size, duration In this case-control study, 12 endometrial samples (eutopic) and 12 endometriotic lesions (ectopic) of women with endometriosis and 12 endometrial control samples were analyzed. Control samples were obtained from women who had no evidence of endometriosis during diagnostic laparoscopy. Control and eutopic endometrial samples were obtained by pipelle. Ectopic samples were obtained during laparoscopy. All women signed the informed consent form and did not receive any hormonal treatments during the last three months. Participants/materials, setting, methods After endometrial tissues collection, gene expression levels of IGF2 and H19 were evaluated using real-time PCR . The occupancy of MeCP2 on two subregions within DMR region of H19 gene was investigated using chromatin immunoprecipitation (ChIP) followed by real-time PCR. One-way ANOVA was used for data analysis. P value less than 0.05 was considered statistically significant. Main results and the role of chance Gene expression profile of H19 and IGF2 was decreased in eutopic and ectopic endometrial lesions of endometriosis group compared with control ones. The decrease in gene expression of H19 in ectopic samples was significant in compared to the control ones while it was nearly significant in compared to the eutopic samples (p = 0.01, p = 0.056, respectively). The ChIP analysis revealed that MeCP2 incorporation in region II (between −3945 and –3818 bp)within DMR region of H19 gene was significantly decreased in eutopic samples compare to control group (p = 0.02) while its decrease was nearly significant in ectopic samples (p = 0.056). However, this DNA methylation profile was not significantly different between eutopic and ectopic endometrial samples in endometriosis in group. Incorporation of MeCP2 in region I (between −2230 and –2103 bp)within DMR region of H19 gene was not significantly different between the eutopic, ectopic and control samples (p &amp;gt; 0.05). (data was presented at 21th Royan International Congress). Limitations, reasons for caution The main limitations of this study is its small sample size. For getting more information, we need to monitor this DNA methylation profile in a large number of women with and without endometriosis. Also, more investigations are required to clarify the further epigenetic modifications in this region. Wider implications of the findings: It seems that reduced expression of IGF2 may be due to hypomethylation of H19-DMR region II while this hypomethylation has no effect on H19 expression in endometriosis. As previously was shown , hypomethylation of H19-DMR causes decrease of IGF2 expression and increase in H19 expression. Trial registration number Not applicable

Study of long non-coding RNA and mitochondrial dysfunction in diabetic rats

Diabetes mellitus (DM) is a worldwide health problem. The Micro- and macro-vascular complications are the major causes of morbidity and mortality of DM. Molecular regulation of mitochondrial fission/fusion cycles is being studied, but the results were not conclusive. The aim of this study is to investigate the possible functional role of lncRNA H19 and its relation to mitofusin-2 (Mfn-2) gene expression in diabetic rats with cardiac and renal complications. Streptozotocin-induced diabetic male, albino rats and a matched control group were investigated. Cardiac weights, blood pressure and ECG were recorded. Biochemical evaluation of cardiac and renal functions was performed. Molecular determination of lncRNA H19 and Mfn-2 gene expression and histological examination by light and electron microscopy for cardiac and renal tissues were performed. Diabetic rats showed a significant increase of left ventricle weight/whole body weight ratio, R wave voltage, and a significant decrease of blood pressure, heart rate, and P wave voltage. At the molecular level, lncRNA H19 and Mfn-2 mRNA showed altered expression with a statistically significant downregulation of Mfn-2 mRNA expression in renal tissues. In conclusion, the changes in lncRNA H19 and Mfn-2 mRNA expression may help better understanding of the pathogenesis of cardiac and renal dysfunctions associated with type 1 DM.

Expression of long noncoding RNA in skin exosomes of patients with vitiligo

Background Vitiligo is a relatively common pigmentation disorder in which the skin melanocytes are lost or destroyed. Experimental downregulation of long noncoding RNA H19 (lnc RNA H19) in keratinocytes promoted melanocytes melanogenesis. lnc RNA H19 acts as an important regulator for micro-RNA let7a (miRNA let7a). miRNA let7a has a powerful role in regulation of cell survival and inducing cell apoptosis.Objectives This study was performed to clarify the suggested role of lnc RNA H19 and miRNA let7a derived from human skin exosomes in vitiligo pathogenesis.Patients and methods The study included 50 patients with vitiligo vulgaris and 50 healthy volunteers serving as controls. From all patients, 4-mm punch skin biopsies were taken. Skin biopsies were examined for lnc RNA H19 and miRNA let7a genes expression using RT-PCR technique.Results The expression of lnc RNA H19 and miRNA let7a in vitiligo lesions (1.736±0.84 and 6.7±2.26 pg/mg, respectively) was significantly higher than their expression in controls (0.8462±0.3483 and 1.514± 0.9202 pg/mg, respectively) (P<0.001). A significant positive correlation was found between the expression of lnc RNA H19 and the disease activity (P=0.0382). Additionally, a positive correlation was detected between the expression of miRNA let7a and the disease duration (P<0.0306).Conclusions We can conclude that upregulation of lnc RNA H19 and miRNA let7a gene expression describes their possible role in the pathogenesis of vitiligo.

A G-quadruplex structure at the 5\u2032 end of the H19 coding region regulates H19 transcription

The H19 gene, one of the best known imprinted genes, encodes a long non-coding RNA that regulates cell proliferation and differentiation. H19 RNA is widely expressed in embryonic tissues, but its expression is restricted in only a few tissues after birth. However, regulation of H19 gene expression remains poorly understood outside the context of genomic imprinting. Here we identified evolutionarily conserved guanine (G)-rich repeated motifs at the 5′ end of the H19 coding region that are consistent with theoretically deduced G-quadruplex sequences. Circular dichroism spectroscopy and electrophoretic mobility shift assays with G-quadruplex-specific ligands revealed that the G-rich motif, located immediately downstream of the transcription start site (TSS), forms a G-quadruplex structure in vitro. By using a series of mutant forms of H19 harboring deletion or G-to-A substitutions, we found that the H19-G-quadruplex regulates H19 gene expression. We further showed that transcription factors Sp1 and E2F1 were associated with the H19-G-quadruplex to either suppress or promote the H19 transcription, respectively. Moreover, H19 expression during differentiation of mouse embryonic stem cells appears to be regulated by a genomic H19 G-quadruplex. These results demonstrate that the G-quadruplex structure immediately downstream of the TSS functions as a novel regulatory element for H19 gene expression.

Evaluation of the Role of Circulating Long Non-Coding RNA H19 as a Promising Novel Biomarker in Plasma of Patients with Gastric Cancer.

H19 is one of the long non-coding RNAs (LncRNA) that is related to the progression of many diseases including cancers. This work was carried out to study the level of the long non-coding RNA; H19, in plasma of patients with gastric cancer (GC) and to assess its significance in their clinical management. Sixty-two participants were enrolled in the present study. The first group included 32 GC patients. The second group was formed of 30 age and sex matched healthy volunteers serving as a control group. Plasma samples were used to assess H19 gene expression using real-time quantitative PCR technique. H19 expression was up-regulated and closely related to TNM cancer stages in GC patients. Using Receiver Operating Characteristic (ROC) curve analysis, a cutoff level of 0.5 was set for H19 expression to diagnose GC cases achieving a sensitivity of 68.75%, specificity of 56.67%, positive predictive value (PPV) 62.86% and negative predictive value (NPV) 62.96% with an area under the curve (AUC) of 72.4%. Combined use of Carcinoembryonic Antigen (CEA) and H19 level in GC diagnosis was evaluated using ROC curve revealing improvement in performance with an area under the curve of 80.4%. Up-regulation of H19 is closely associated with gastric cancer displaying progressive up-regulation in advanced stages of the disease implementing its role as a potential non-invasive diagnostic biomarker in gastric cancer and as a novel tool in gastric cancer management with better performance achieved on using both CEA and H19 simultaneously.

Alterations in expression of imprinted genes from the H19/IGF2 loci in a multigenerational model of intrauterine growth restriction (IUGR)

The H19/IGF2 imprinted loci have attracted recent attention because of their role in cellular differentiation and proliferation, heritable gene regulation, and in utero or early postnatal growth and development. Expression from the imprinted H19/IGF2 locus involves a complex interplay of 3 means of epigenetic regulation: proper establishment of DNA methylation, promoter occupancy of CTCF, and expression of microRNA-675. We have demonstrated previously in a multigenerational rat model of intrauterine growth restriction the epigenetic heritability of adult metabolic syndrome in a F2 generation. We have further demonstrated abrogation of the F2 adult metabolic syndrome phenotype with essential nutrient supplementation of intermediates along the 1-carbon pathway and shown that alterations in the metabolome precede the adult onset of metabolic syndrome. The upstream molecular and epigenomic mediators underlying these observations, however, have yet to be elucidated fully. In the current study, we sought to characterize the impact of the intrauterine growth-restricted lineage and essential nutrient supplementation on both levels and molecular mediators of H19 and IGF2 gene expression in the F2 generation. F2 intrauterine growth-restricted and sham lineages were obtained by exposing P1 (grandmaternal) pregnant dams to bilateral uterine artery ligation or sham surgery at gestational day 19.5. F1 pups were allocated to the essential nutrient supplemented or control diet at postnatal day 21, and bred at 6-7 weeks of age. Hepatic tissues from the resultant F2 offspring at birth and at weaning (day 21) were obtained. Bisulfite modification and sequencing was employed for methylation analysis. H19 and IGF2 expression was measured by quantitative polymerase chain reaction. Promoter occupancy was quantified by the useof chromatin immunoprecipitation, or ChIP, against CTCF insulator proteins. Growth-restricted F2 on control diet demonstrated significant down-regulation in H19 expression compared with sham lineage (0.7831 vs 1.287; P < .05); however, essential nutrient supplementation diet abrogates this difference (4.995 vs 5.100; P > .05). Conversely, Igf2 was up-regulated by essential nutrient supplemented diet on the sham lineage (2.0 fold, P = .01), an effect that was not observed in the growth restricted offspring. A significant differential methylation was observed inthe promoter region of region H19 among the intrauterine growth-restricted lineage (18% vs 25%; P < .05) on a control diet, whereas the essential nutrient supplemented diet was alternately associated with hypermethylation in both lineages (sham: 50%; intrauterine growth restriction: 84%, P < .05). Consistent with essential nutrient supplementation impacting the epigenome, a decrease of CTCF promoter occupancy was observed in CTCF4 of the growth restricted lineage (2.45% vs 0.56%; P < .05) on the control diet, an effect that was repressed with essential nutrient supplementation. Heritable growth restriction is associated with changes in H19 gene expression; these changes are reversible with diet supplementation to favorably impact adult metabolic syndrome.

Altered gene expression of H19 and IGF2 in placentas from ART pregnancies

IntroductionThe aim of this study is to determine whether the gene expression and associated DNA methylation regulation of H19 and IGF2 are altered in placentas conceived by assisted reproductive technologies (ART) compared to natural conceptions. Methods113 pregnancies were recruited resulting in 119 placentas (83 singletons and 36 twins), where 56 were conceived via in vitro fertilization (IVF), 41 via intracytoplasmic sperm injection (ICSI), and 22 naturally. Regulation of imprinting of H19 and IGF2 was determined by the DNA methylation status at three CpG sites within the H19 imprinting control region 1 (ICR1) using bisulphite pyrosequencing. Expression of H19 and IGF2 in 45 of these placentas (17 IVF, 14 ICSI, and 14 NC) was measured by determining the relative mRNA transcript levels using RT-qPCR in placental villi. ResultsPlacental weight and birth weight were not significantly different between groups. H19 expression was significantly increased in both IVF and ICSI placentas when compared to controls (1.8 and 1.9 fold higher, respectively). Conversely, IGF2 was significantly decreased in both ART groups (0.8 and 0.7 fold lower, respectively). Mean DNA methylation at ICR1 was found to be similar between all groups. No correlation was found between DNA methylation at ICR1 and expression of either gene. However, a significant inverse relationship was found between H19 and IGF2 expression. ConclusionWe provide evidence of altered H19 and IGF2 expression in ART placentas. The altered expression pattern may suggest a loss of imprinting on the paternal allele. Furthermore, these alterations may not be entirely associated with DNA methylation at ICR1. We show further indirect evidence of the H19-IGF2 inverse expression pattern.

Faithful expression of imprinted genes in donor cells of SCNT cloned pigs

To understand if the genomic imprinting status of the donor cells is altered during the process of SCNT (somatic cell nuclear transfer), cloned pigs were produced by SCNT using PEF (porcine embryonic fibroblast) and P-PEF (parthenogenetic-PEF) cells as donors. Then, the gene expression and methylation patterns of H19, IGF2, NNAT and MEST were compared between PEF vs. C-PEF (cloned-PEF), P-PEF vs. CP-PEF (cloned-P-PEF), respectively. Taken together, the results revealed that there was no significant difference in the expression of imprinted genes and conserved genomic imprints between the donor and cloned cells.

Estrogen regulates luminal progenitor cell differentiation through H19 gene expression.

Although the role of estrogen signaling in breast cancer development has been extensively studied, the mechanisms that regulate the indispensable role of estrogen in normal mammary gland development have not been well studied. Because of the unavailability of culture system to maintain estrogen-receptor-positive (ERα+) cells in vitro, the molecular mechanisms that regulate estrogen/ERα signaling in the normal human breast are unknown. In the present study, we examined the effects of estrogen signaling on ERα+ human luminal progenitors using a modified matrigel assay and found that estrogen signaling increased the expansion potential of these progenitors. Furthermore, we found that blocking ERα attenuated luminal progenitor expansion and decreased the luminal colony-forming potential of these progenitors. Additionally, blocking ERα decreased H19 expression in the luminal progenitors and led to the development of smaller luminal colonies. We further showed that knocking down the H19 gene in the luminal progenitors significantly decreased the colony-forming potential of the luminal progenitors, and this phenotype could not be rescued by the addition of estrogen. Lastly, we explored the clinical relevance of the estrogen–H19 signaling axis in breast tumors and found that ERα+ tumors exhibited a higher expression of H19 as compared with ERα− tumors and that H19 expression showed a positive correlation with ERα expression in those tumors. Taken together, the present results indicate that the estrogen–ERα–H19 signaling axis plays a role in regulating the proliferation and differentiation potentials of the normal luminal progenitors and that this signaling network may also be important in the development of ER+ breast cancer tumors.

H19 and MEST gene expression and histone modification in blastocysts cultured from vitrified and fresh two-cell mouse embryos

Vitrification of embryos is a routine procedure in many IVF laboratories. The effect of vitrification on gene expression and some modifications of H3 histone in H19 and MEST imprinted genes in blastocysts produced in vitro from non-vitrified and vitrified two-cell embryos was investigated. The expression level of the chosen imprinted genes increased significantly (P < 0.05) in experimental groups compared with in-vivo blastocysts (control group). H3K9me2 decreased, whereas H3K9ac increased in the experimental group compared with the control group. The increases in the expression levels of the imprinted genes, and the attendant changes in histone and chromatin status associated with in-vitro culture of embryos from the two-cell stage, are unaffected by prior vitrification and warming. In the present study, it was shown that such changes are solely caused by the effect of in-vitro culture, irrespective of vitrification. Although these genes are sensitive to environmental changes, vitrification seems to have no additional effect on these genes and on the histone marks, and can threfore be considered to be a process with minimum damage for embryo cryopreservation in assisted reproductive technology applications.

Analysis of imprinted IGF2/H19 gene methylation and expression in normal fertilized and parthenogenetic embryonic stem cells of pigs

To determine whether the genomic imprinting can be maintained during the process of embryonic stem (ES) cell derivation from pig blastocysts, mRNA and DNA methylation at the IGF2/H19 imprinting control region in putative ES cells derived from in vitro fertilized (IVF) and parthenogenetic (PG) embryos were investigated. In the present study, one IVF- and three PG ES-like cell lines were established and analyzed for cellular characteristics such as pluripotent marker expression and differentiation capacity. The results showed that these putative ES cells derived from pig blastocysts fulfilled the general “stemness” criteria. The expression of the H19 gene was significantly greater in PG blastocysts than IVF blastocysts, but there were greater amounts of IGF2 in IVF than PG blastocysts. Of these putative ES cell lines, one PG line had less H19 gene expression than a IVF ES cell line while the other two PG lines had much greater expression of the H19 gene than the IVF line. In contrast, the IGF2 gene was upregulated in the same PG cell line relative to the other two PG cell lines and transcript abundance was similar to IVF ES-like cells. Despite the variable amounts of mRNA among the PG cell lines, the IGF2/H19 gene had a differentially methylated region (DMR) 3 was typically un-methylated in all PG cells, and hemi-methylated in the IVF cells. These findings indicated that the mRNA of H19 and IGF2 genes is susceptible to in vitro environments during the process of ES cell derivation from blastocysts but DNA methylation status at this region was well maintained. These altered gene expressions may not be associated with the methylation of the imprinting control region at this locus. Therefore, with their uni-parental genotype, the pluripotent differentiation potentials of PG ES cells could be a valuable tool for understanding genomic imprinting in embryonic development.