Effect of triazophos on the expression of H19 and Sox2OT genes in Danio rerio

Expression of H19 and Igf2 genes in uniparental mouse ES cells during in vitro and in vivo differentiation

Low H19 and abundant IGF-II expression may have a role in the development of adrenocortical carcinomas. In the mouse, the H19 promoter area has been found to be methylated when transcription of the H19 gene is silent and unmethylated when it is active. We used PCR-based methylation analysis and bisulfite genomic sequencing to study the cytosine methylation status of the H19 promoter region in 16 normal adrenals and 30 pathological adrenocortical samples. PCR-based analysis showed higher methylation status at three HpaII-cutting CpG sites of the H19 promoter in adrenocortical carcinomas and in a virilizing adenoma than in their adjacent normal adrenal tissues. Bisulfite genomic sequencing revealed a significantly higher mean degree of methylation at each of 12 CpG sites of the H19 promoter in adrenocortical carcinomas than in normal adrenals (P < 0.01 for all sites) or adrenocortical adenomas (P < 0.01, except P < 0.05 for site 12 and P > 0.05 for site 11). The mean methylation degree of the 12 CpG sites was significantly higher in the adrenocortical carcinomas (mean +/- SE, 76 +/- 7%) than in normal adrenals (41 +/- 2%) or adrenocortical adenomas (45 +/- 3%; both P < 0.005). RNA analysis indicated that the adrenocortical carcinomas expressed less H19 but more IGF-II RNAs than normal adrenal tissues did. The mean methylation degree of the 12 H19 promoter CpG sites correlated negatively with H19 RNA levels (r = -0.550; P < 0.01), but positively with IGF-II mRNA levels (r = 0.805; P < 0.001). In the adrenocortical carcinoma cell line NCI-H295R, abundant IGF-II, but minimal H19, RNA expression was detected by Northern blotting. Treatment with a cytosine methylation inhibitor, 5-aza-2'-deoxycytidine, increased H19 RNA expression, whereas it decreased IGF-II mRNA accumulation dose- and time-dependently (both P < 0.005) and reduced cell proliferation to 10% in 7 d. Our results suggest that altered DNA methylation of the H19 promoter is involved in the abnormal expression of both H19 and IGF-II genes in human adrenocortical carcinomas.

Low H19 and abundant IGF-II expression may have a role in the development of adrenocortical carcinomas. In the mouse, the H19 promoter area has been found to be methylated when transcription of the H19 gene is silent and unmethylated when it is active. We used PCR-based methylation analysis and bisulfite genomic sequencing to study the cytosine methylation status of the H19 promoter region in 16 normal adrenals and 30 pathological adrenocortical samples. PCR-based analysis showed higher methylation status at three HpaII-cutting CpG sites of the H19 promoter in adrenocortical carcinomas and in a virilizing adenoma than in their adjacent normal adrenal tissues. Bisulfite genomic sequencing revealed a significantly higher mean degree of methylation at each of 12 CpG sites of the H19 promoter in adrenocortical carcinomas than in normal adrenals (P < 0.01 for all sites) or adrenocortical adenomas (P < 0.01, except P < 0.05 for site 12 and P > 0.05 for site 11). The mean methylation degree of the 12 CpG sites was significantly higher in the adrenocortical carcinomas (mean ± se, 76 ± 7%) than in normal adrenals (41 ± 2%) or adrenocortical adenomas (45 ± 3%; both P < 0.005). RNA analysis indicated that the adrenocortical carcinomas expressed less H19 but more IGF-II RNAs than normal adrenal tissues did. The mean methylation degree of the 12 H19 promoter CpG sites correlated negatively with H19 RNA levels (r = −0.550; P < 0.01), but positively with IGF-II mRNA levels (r = 0.805; P < 0.001). In the adrenocortical carcinoma cell line NCI-H295R, abundant IGF-II, but minimal H19, RNA expression was detected by Northern blotting. Treatment with a cytosine methylation inhibitor, 5-aza-2′-deoxycytidine, increased H19 RNA expression, whereas it decreased IGF-II mRNA accumulation dose- and time-dependently (both P < 0.005) and reduced cell proliferation to 10% in 7 d. Our results suggest that altered DNA methylation of the H19 promoter is involved in the abnormal expression of both H19 and IGF-II genes in human adrenocortical carcinomas.

Dissociation of IGF2 and H19 imprinting in human brain

The H19 gene is a parenterally imprinted maternally expressed gene which has a pivotal role in embryogenesis and fetal development. It is tightly linked to the IGF-II gene on chromosome 11p15.5 which is reciprocally imprinted. We studied the expression of the human H19 by in situ hybridization in an embryo 35 days post coitus (dpc) and in a fetus from the second trimester of pregnancy. The expression pattern of H19 in the human fetal tissues was similar to its expression in the mouse, and paralleled, with some exceptions, the expression of IGF-II in human fetuses. Abundant expression was found in organs comprising the fetoplacental unit: the placenta, the fetal adrenal, and liver. The expression in the fetal adrenal cortex was most prominent in the definitive cortex and somewhat weaker in the fetal zone. Considerable expression of H19 was found in the fetal liver as early as 35 dpc and in the second trimester. Hematopoietic cells in fetal liver did not express the gene. Moderate expression of H19 was detected in the epithelium of the small intestines, in endometrial stroma and Fallopian tube. In the kidney conspicuous labeling of the metanephric blastema was noted, which was markedly reduced with differentiation to tubules. This pattern of expression is identical to that of IGF-II in the fetal kidney and is relevant to the evolution of Wilms' tumor. No expression of H19 was found in the neural tube of the first trimester embryo or in the developing fetal brain in the second trimester, nor were transcripts detected in the choroid plexus.(ABSTRACT TRUNCATED AT 250 WORDS)

The expression of H19 and insulin-like growth factor-II (IGF-II) genes is important for fetal growth, and the misexpression of these genes may also be involved in the development of some tumors. In human fetal adrenals, H19 and IGF-II expression levels are very high. We show here that H19 is strongly expressed (approximately 50% of the expression in fetal adrenals and 6-fold higher than that in adult liver) in normal adult adrenals (n = 9), adrenocortical adenomas (n = 28), and hyperplastic adrenals (n = 11). In four hormonally active adrenocortical carcinomas, very low levels of H19 ribonucleic acid (RNA) were detected, whereas IGF-II was highly expressed. In cultured adrenocortical cells, ACTH, (Bu)2cAMP, and cholera toxin increased H19 RNA accumulation 2- to 5-fold (P < 0.01), but had no significant effect on IGF-II messenger RNA levels. In pheochromocytomas (n = 22), H19 expression was variable, on the average, about 13% of the expression in the adjacent adrenal cortex. In primary cultures of pheochromocytoma cells, H19 RNA was not detectable via Northern blot analysis. Our data show that H19 expression is maintained at high levels in adult human adrenals and benign neoplasms. H19 RNA is up-regulated by ACTH in adult adrenocortical cells. The very low levels of H19 expression in hormonally active adrenocortical carcinomas suggest that loss of H19 expression may be associated with malignancy in these neoplasms.

Why is the female population more susceptible to cholestasis-induced liver injury-Could it be long noncoding RNA H19?

Monoallelic expression of the imprinted H19 and insulin-like growth factor-2 (Igf2) genes depends on the hypomethylation of the maternal allele and hypermethylation of the paternal allele of the H19 upstream region. Previous studies from our laboratory on liver carcinogenesis in the F1 hybrid of Fischer 344 (F344) and Sprague-Dawley Alb SV40 T Ag transgenic rat (SD) strains revealed the biallelic expression of H19 in hepatomas. We undertook a comparative study of the DNA methylation status of the upstream region of H19 in fetal, adult, and neoplastic liver. Bisulfite DNA sequencing analysis of a 3.745-kb DNA segment extending from 2950 to 6695 bp of the H19 upstream region revealed marked variations in the methylation patterns in fetal, adult, and neoplastic liver. In the fetal liver, equal proportions of hyper- and hypomethylated strands revealed the differentially methylated status of the parental alleles, but in neoplastic liver a pronounced change in the pattern of methylation was observed with a distinct change to hypomethylation in the short segments between 2984 and 3301 bp, 6033-6123 bp, and 6518-6548 bp. These results indicated that methylation of all cytosines in this region may contribute to the imprinting status of the rat H19 gene. This phenomenon of differential methylation-related epigenetic alteration in the key cis-regulatory domains of the H19 promoter influences switching to biallelic expression in hepatocellular carcinogenesis. Similar to mouse and human, we showed that the zinc-finger CCTCC binding factor (CTCF) binds to the unmethylated CTCF binding site in the upstream region to influence monoallelic imprinted expression in fetal liver. CTCF does not appear to be rate limiting in fetal, normal, and neoplastic liver. 3' to the CTCF binding sites, another DNA region exhibits methylation of CpG's in both DNA strands in adult liver, retention of the imprint in fetal liver, and complete demethylation in neoplastic liver. In this region is also a putative binding site for a basic helix-loop-helix leucine-zipper transcription factor, TFEB. The differential CpG methylation seen in the adult that involves the TFEB binding site may explain the lack of expression of the H19 gene in adult normal liver. Furthermore, these findings demonstrate that the loss of imprinting of the H19 gene in hepatic neoplasms of the SD Alb SV40 T Ag transgenic rat is directly correlated with and probably the result of differential methylation of CpG dinucleotides in two distinct regions of the gene that are within 4 kb 5' of the transcription start site. Cytogenetic analysis of hepatocytes in the transgenic animal prior to the appearance of nodules or neoplasms indicates a role of such loss of imprinting in the very early period of neoplastic development, possibly the transition from the stage of promotion to that of progression.

Study question Does re-vitrification affect on methylation level of H19/IGF2 DMR differentially methylated region(DMR) in human in vitro-produced blastocysts? Summary answer The results of this study showed that re-vitrification had no significant effect on the methylation level of H19 / IGF2 DMR region in human blastocysts. What is known already Nowadays, re-vitrification seems to be a successful and useful method to prevent the discarding of extra embryos. Several reports have indicated that the embryo manipulations during ART may result in epigenetic errors. This has been attributed to the concurrency of the ART manipulations and epigenetic events after fertilization. The effect of embryo re-vitrification on epigenetic changes has not been studied so far. The aim of this study is to evaluate the effect of embryo re-vitrification on methylation level of H19/IGF2 differentially methylated region (DMR) in donated human blastocysts. Study design, size, duration The effect of re-vitrification on methylation level of H19/IGF2 DMR was evaluated in 9 ICSI-derived human embryos individually and the expressions of H19 and IGF2 were assessed in 15 embryos individually. Embryos were donated from fertile couples referring for family balancing program. Donated embryos were cultured to blastocyst stage and high quality blastocyst (AA-AB) assigned to three groups: fresh, vitrified and re-vitrification. Scoring of blastocysts were according to Gardner et al. (2012) grading system. Participants/materials, setting, methods Collected blastocysts were vitrified on Cryotech carriers, with the method described by Kuwayama. After warming of blastocysts and 4-5 hours culturing of them, expression of H19 and IGF2 genes in blastocysts evaluated with Real time PCR and methylation levels of H19/IGF2 DMR in blastocysts were analyzed by Bisulfite Sequencing PCR (BSP) technique. 10 clones were sequenced per replicate. Methylation status of lymphocyte was used as control to verify the credibility of our analysis system. Main results and the role of chance The results showed that the overall percentage of methylated CpGs of H19/IGF2 DMR in the control, vitrification, re-vitrification groups and lymphocyte were 42.71, 40.90, 39.23% and 49.30 %. respectively. There was no significant difference in methylation level of H19/IGF2 DMR between the groups. H19 and IGF2 expression did not show a significant difference in vitrified group compared to the control and in re-vitrified group compared to vitrified. Limitations, reasons for caution - Small number of samples examined due to difficult access to human blastocysts donated by fertile couples. - Evaluation of high-quality embryos donated by healthy fertile couples while embryos of infertile couples may respond differently. Wider implications of the findings According to the obtained results, embryo re-vitrification did not have a significant change on the methylation level of H19/IGF2 DMR and expression of H19 and IGF2 genes. However, that further studies on the effect of re-vitrification on epigenetic errors in low-quality embryos and the embryos of infertile couples are needed. Trial registration number not applicable

Effect of acute exposure of triazophos on histological structure and apoptosis of the brain and liver of zebrafish (Danio rerio)

Loss of allele-specific expression by the imprinted genes IGF2 and H19 has been correlated with a differentially methylated region (DMR) upstream to the H19 gene. The H19-DMR contains seven potential CCCTC-binding factor (CTCF) binding sites. CTCF is a chromatin insulator and a multifunctional transcription factor whose binding to the H19-DMR is suppressed by DNA methylation. Our study included a group of 41 head and neck squamous cell carcinoma (HNSCC) samples. The imprinting status of the H19 gene was analyzed in 11 out of 35 positive cases for H19 gene expression, and only 1 of them showed loss of imprinting. We detected a significant correlation (P = 0.041, Fisher's exact test) between H19 expression and tumor recurrence. Among H19 positive cases, six were T2, in which five developed recurrence and/or metastasis. Inversely, in the group of tumors that showed no H19 gene expression, 5 out of 24 were T2 and only 1 presented regional recurrence. These data support the hypothesis that H19 expression could be used as a prognostic marker to indicate recurrence in early stage tumors. We also examined the methylation of the CTCF binding site 1 in a subgroup of these samples. The H19 gene silencing and loss of imprinting were not correlated with the methylation pattern of the CTCF binding site 1. However, the significant correlation between H19 expression and tumor recurrence suggest that this transcript could be a marker for the progression of HNSCC.

We examined H19 and insulin-like growth factor 2 (IGF2) gene expression in normal endometrium (12 cases), hyperplasia (27 cases), and cancer (27 cases) by non-radioactive in situ hybridization. H19 was not expressed in the epithelium of normal endometrium, but its frequency of expression was 15% in hyperplastic and 60% in neoplastic epithelium. In stroma cells, H19 frequency of expression was 75% in normal endometrium, 55% in hyperplasia, and 37% in carcinoma. According to the grade of endometrial cancer cell differentiation, H19 showed increased frequency and level of expression in the epithelium from well to moderately and poorly differentiated tissues. Our results indicate that H19 expression in epithelial cells of endometrial hyperplasia and cancer merits further investigation and could be useful as a complementary histopathologic and prognostic marker among other modalities in endometrial cancer. IGF2 expression did not appear useful for diagnostic or prognostic purposes.

We examined H19 and insulin-like growth factor 2 (IGF2) gene expression in normal endometrium (12 cases), hyperplasia (27 cases), and cancer (27 cases) by non-radioactivein situhybridization. H19 was not expressed in the epithelium of normal endometrium, but its frequency of expression was 15% in hyperplastic and 60% in neoplastic epithelium. In stroma cells, H19 frequency of expression was 75% in normal endometrium, 55% in hyperplasia, and 37% in carcinoma. According to the grade of endometrial cancer cell differentiation, H19 showed increased frequency and level of expression in the epithelium from well to moderately and poorly differentiated tissues. Our results indicate that H19 expression in epithelial cells of endometrial hyperplasia and cancer merits further investigation and could be useful as a complementary histopathologic and prognostic marker among other modalities in endometrial cancer. IGF2 expression did not appear useful for diagnostic or prognostic purposes.

Biallelic expression of the H19 and IGF2 genes in hepatocellular carcinoma

Effects of UV filters (benzophenones and octocrylene) and mifepristone on different life stages of zebrafish (Danio rerio)