Dlk1-Dio3 Region Research Articles

Epigenetic Regulation of DLK1-DIO3 Region in Thyroid Carcinoma.

Non-coding RNAs (ncRNAs) have emerged as pivotal regulators in cellular biology, dispelling their former perception as 'junk transcripts'. Notably, the DLK1-DIO3 region harbors numerous ncRNAs, including long non-coding RNAs (lncRNAs) and over 50 microRNA genes. While papillary thyroid cancer showcases a pervasive decrease in DLK1-DIO3-derived ncRNA expression, the precise mechanisms driving this alteration remain elusive. We hypothesized that epigenetic alterations underlie shifts in ncRNA expression during thyroid cancer initiation and progression. This study aimed to elucidate the epigenetic mechanisms governing DLK1-DIO3 region expression in this malignancy. We have combined the analysis of DNA methylation by bisulfite sequencing together with that of histone modifications through ChIP-qPCR to gain insights into the epigenetic contribution to thyroid cancer in cell lines representing malignancies with different genetic backgrounds. Our findings characterize the region's epigenetic signature in thyroid cancer, uncovering distinctive DNA methylation patterns, particularly within CpG islands on the lncRNA MEG3-DMR, which potentially account for its downregulation in tumors. Pharmacological intervention targeting DNA methylation combined with histone deacetylation restored ncRNA expression. These results contribute to the understanding of the epigenetic mechanisms controlling the DLK1-DIO3 region in thyroid cancer, highlighting the combined role of DNA methylation and histone marks in regulating the locus' expression.

Identification of the porcine IG-DMR and abnormal imprinting of DLK1-DIO3 in cloned pigs.

Correct reprogramming of the DLK1-DIO3 imprinted region is critical for the development of cloned animals. However, in pigs, the imprinting and regulation of the DLK1-DIO3 region has not been systematically analyzed. The objective of this study was to investigate the imprinting status and methylation regulation of the DLK1-DIO3 region in wild-type and cloned neonatal pigs. We mapped the imprinting control region, IG-DMR, by homologous alignment and validated it in sperm, oocytes, fibroblasts, and parthenogenetic embryos. Subsequently, single nucleotide polymorphism-based sequencing and bisulfite sequencing polymerase chain reaction were conducted to analyze imprinting and methylation in different types of fibroblasts, as well as wild-type and cloned neonatal pigs. The results showed that Somatic cell nuclear transfer (SCNT) resulted in hypermethylation of the IG-DMR and aberrant gene expression in the DLK1-DIO3 region. Similar to wild-type pigs, imprinted expression and methylation were observed in the surviving cloned pigs, whereas in dead cloned pigs, the IG-DMR was hypermethylated and the expression of GTL2 was nearly undetectable. Our study reveals that abnormal imprinting of the DLK1-DIO3 region occurs in cloned pigs, which provides a theoretical basis for improving the cloning efficiency by gene editing to correct abnormal imprinting.

Involvement of PGC7 and UHRF1 in the regulation of DNA methylation of the IG-DMR in the imprinted Dlk1-Dio3 locus.

The gene dosage at the imprinted Dlk1-Dio3 locus is critical for cell growth and development. A relatively high gene expression within the Dlk1-Dio3 region, especially the active expression of Gtl2, has been identified as the only reliable marker for cell pluripotency. The DNA methylation state of the IG-DNA methylated regions (DMR), which is located upstream of the Gtl2 gene, dominantly contributes to the control of gene expression in the Dlk1-Dio3 locus. However, the precise mechanism underlying the regulation of DNA methylation in the IG-DMR remains largely unknown. Here, we use the F9 embryonal carcinoma cell line, a low pluripotent cell model, to identify the mechanism responsible for DNA methylation in the IG-DMR, and find that the interaction of PGC7 with UHRF1 is involved in maintaining DNA methylation and inducing DNA hypermethylation in the IG-DMR region. PGC7 and UHRF1 cooperatively bind in the IG-DMR to regulate the methylation of DNA and histones in this imprinted region. PGC7 promotes the recruitment of DNMT1 by UHRF1 to maintain DNA methylation in the IG-DMR locus. The interaction between PGC7 and UHRF1 strengthens their binding to H3K9me3 and leads to further enrichment of H3K9me3 in the IG-DMR by recruiting the specific histone methyltransferase SETDB1. Consequently, the abundance of H3K9me3 promotes DNMT3A to bind to the IG-DMR and increases DNA methylation level in this region. In summary, we propose a new mechanism of DNA methylation regulation in the IG-DMR locus and provide further insight into the understanding of the difference in Gtl2 expression levels between high and low pluripotent cells.

Emerging Evidence of the Functional Impact of the miR379/miR656 Cluster (C14MC) in Breast Cancer.

Many microRNAs exist in clusters that share comparable sequence homology and may target genes in a common pathway. The miR-379/miR-656 (C14MC) cluster is imprinted in the DLK1-Dio3 region of 14q32.3 and contains 42 miRNAs. It plays a functional role in numerous biological pathways including vascular remodeling and early development. With many C14MC miRNAs highlighted as potential tumor suppressors in a variety of cancers, the role of this cluster in breast cancer (BC) has garnered increased attention in recent years. This review focuses on C14MC in BC, providing an overview of the constituent miRNAs and addressing each in terms of functional impact, potential target genes/pathways, and, where relevant, biomarker capacity. Studies have revealed the regulation of key factors in disease progression and metastasis including tyrosine kinase pathways and factors critical to epithelial–mesenchymal transition (EMT). This has potentially important clinical implications, with EMT playing a critical role in BC metastasis and tyrosine kinase inhibitors (TKIs) in widespread use for the treatment of BC. While the majority of studies have reported tumor-suppressing roles for these miRNAs, some have highlighted their potential as oncomiRs. Understanding the collective contribution of miRNAs within C14MC to BC may support improved understanding of disease etiology and present novel approaches to targeted therapy.

A MicroRNA Cluster in the DLK1-DIO3 Imprinted Region on Chromosome 14q32.2 Is Dysregulated in Metastatic Hepatoblastomas.

Hepatoblastoma (HB) is the most common malignant liver neoplasm in children. Despite progress in HB therapy, outcomes for patients with metastatic disease remain poor. Dysregulation of miRNA expression is one of the potential epigenetic mechanisms associated with pathogenesis of HB. However, miRNA profiles related to the different stages of HB tissues and cells, in particular of lung metastatic tumor cells, are unknown. In the present study, using array-based miRNA expression and DNA methylation analysis on formalin-fixed paraffin-embedded tissues, we aimed to identify miRNA changes that can discriminate between lung metastatic tumors, primary tumors (fetal and embryonal subtypes), and nontumorous surrounding livers. Our analysis demonstrated that a large cluster of microRNAs and snoRNAs located within the 14q32.2 DLK1-DIO3 region showed a strikingly upregulated expression pattern in HB tumors, especially metastatic tumors, compared to normal liver tissues. This revealed dysregulation of miRNAs similar to that seen in a malignant stem-like subtype of hepatocellular carcinoma associated with poor prognosis. These findings in HB mirror similar findings made in multiple other cancer types. With further analysis this may in future allow stratification of different stages and types of HB tumors based on their miRNA profiles, which could lead to new approaches to diagnosis and treatment in progressive HB patients.

Deregulated expression of the imprinted DLK1-DIO3 region in glioblastoma stemlike cells: tumor suppressor role of lncRNA MEG3.

BackgroundGlioblastoma (GBM) stemlike cells (GSCs) are thought to be responsible for the maintenance and aggressiveness of GBM, the most common primary brain tumor in adults. This study aims at elucidating the involvement of deregulations within the imprinted delta-like homolog 1 gene‒type III iodothyronine deiodinase gene (DLK-DIO3) region on chromosome 14q32 in GBM pathogenesis.MethodsReal-time PCR analyses were performed on GSCs and GBM tissues. Methylation analyses, gene expression, and reverse-phase protein array profiles were used to investigate the tumor suppressor function of the maternally expressed 3 gene (MEG3).ResultsLoss of expression of genes and noncoding RNAs within the DLK1-DIO3 region was observed in GSCs and GBM tissues compared with normal brain. This downregulation is mainly mediated by epigenetic silencing. Kaplan–Meier analysis indicated that low expression of MEG3 and MEG8 long noncoding (lnc)RNAs significantly correlated with short survival in GBM patients. MEG3 restoration impairs tumorigenic abilities of GSCs in vitro by inhibiting cell growth, migration, and colony formation and decreases in vivo tumor growth, reducing infiltrative growth. These effects were associated with modulation of genes involved in cell adhesion and epithelial-to-mesenchymal transition (EMT).ConclusionIn GBM, MEG3 acts as a tumor suppressor mainly regulating cell adhesion, EMT, and cell proliferation, thus providing a potential candidate for novel GBM therapies.

Construction of constitutive expression systems of microRNAs from the genomic region DLK1-DIO3 with putative tumor suppressor role

Thyroid carcinoma is the most common endocrine cancer in the world. Over the last years attention has been focused on the role of microRNAs (miRs), small non-coding RNAs molecules, in the tumoral thyroid cell biology, especially in the diagnosis and prognosis of the papillary histotype (PTC). In a recent study of the group, the expression of miRs from the DLK1-DIO3 genomic region has been shown to be decreased in PTC. Importantly, distinct members from this genomic region have already been described as tumor suppressor miRs in several types of cancer. The aim of the project herein was to identify, based on target prediction and gene enrichment analysis, candidate miRs with tumor suppressor potential and to generate DNA plasmid constructions. MiR-495-3p and miR-485-5p were identified as the candidates with the highest tumor suppressor potential role in PTC among more than 50 miRs from the DLK1-DIO3 region. The construction of expression systems containing the miRs was performed based in mollecular clonning methodology and will certainly contribute to further functional validation of the role of these miRs in PTC.

Relationship between Altered Gene Expression and DNA Methylation of the DLK1-DIO3 region in Azacitidine-Treated Patients with Myelodysplastic Syndromes and Acute Myeloid Leukemia with Myelodysplasia-Related Changes

INTRODUCTIONMyelodysplastic syndromes (MDS) represent a heterogeneous spectrum of hematopoietic stem cell disorders characterized by inefficient hematopoiesis, peripheral blood cytopenia, dysplasia in one or more myeloid cell lineages, and a tendency to evolve into acute myeloid leukemia (AML). Hypomethylating agent azacitidine (AZA) is a standard therapy for patients with higher-risk MDS and AML with myelodysplasia-related changes (AML-MRC).The DLK1-DIO3 region is located on chromosome 14q32 and contains one of the largest miRNA clusters in the genome. Functions of these miRNAs have been linked to apoptosis and proliferation. Their expression is under the control of a differentially methylated region (DMR) located in the promoter of MEG3 gene. Significant overexpression of these miRNAs has previously been found in MDS and acute promyelocytic leukemia (APL). In APL, this upregulation has been linked to hypermethylation of MEG3-DMR. AIMSWe examined (i) expression of the 14q32 miRNAs and mRNAs (DLK1, RTL1, DIO3, MEG3, and MEG8) and (ii) DNA methylation of MEG3-DMR in patients with higher-risk MDS and AML-MRC before and during AZA treatment with the aim to reveal whether the overexpression of these miRNAs is epigenetically regulated in myelodysplasia. METHODSThe study comprised 24 paired samples of CD34+ bone marrow cells obtained from 12 patients with higher-risk MDS and AML-MRC before and during AZA therapy. miRNA profiling was done using Agilent microarrays and mRNA levels of the genes located within the 14q32 region were assessed by Illumina microarrays. Measurement of MEG3 level by RT-qPCR confirmed the microarray results on an independent cohort (81 MDS and 14 AML-MRC patients, and 13 controls). Methylation status of MEG3 promoter was investigated by amplicon bisulfite sequencing. RESULTSWe studied levels of miRNAs and mRNAs encoded in the DLK1-DIO3 region and found that 50 % of untreated patients showed strong upregulation of expression activity within the locus. Following AZA therapy, we observed a significant reduction in the miRNA/mRNA expression to nearly normal levels, suggesting that expression activity might be affected by hypomethylation. Interestingly, expression levels of miRNAs and mRNAs positively correlated, indicating that transcriptional activity was regulated uniformly throughout the region.Evaluation of expression data with clinical features in the pretreatment samples revealed that expression activity was related to BM blast count, patient diagnosis, and outcome. High expression was significantly associated with the diagnosis of AML-MRC and poor outcome, whereas low expression with MDS and favorable outcome. No chromosomal abnormality or cytogenetic risk category was linked to the expression changes.In the patients before treatment, we revealed a significant hypermethylation of a part of the MEG3-DMR that closely preceded the MEG3 start codon. This hypermethylation was seen in 50 % of the patients and negatively correlated with expression activity. Interestingly, the hypermethylation was associated with longer progression-free survival after initiation of AZA therapy (hazard ratio = 0.220, P = 0.03).Following AZA therapy, we identified an apparent decrease of methylation in another part of MEG3-DMR. This hypomethylation might be attributed to the hypomethylating effect of AZA and could therefore influence the deregulated expression of the miRNA cluster.Finally, we investigated the potential application of the deregulation observed in this locus for prognostic purposes regarding the responsiveness to AZA treatment. Although, we identified changes in expression and methylation levels before AZA therapy, these changes were not associated with future response status. CONLUSIONSHere, we correlated changes in expression of 14q32 miRNAs and mRNAs with specific methylation changes in its regulatory sequences in higher-risk MDS and AML-MRC patients treated with AZA. We thoroughly investigated a complex regulatory mechanism within this region and based on our data, we assumed that epigenetic alterations affect expression activity in the locus. However, also other mechanisms such as influence of some transcription factors may play yet unrecognized roles in the resulting state.Supported by grants 17-31398A, 16-33617A, 16-33485A, and 00023736 (Ministry of Health of the Czech Republic). DisclosuresNo relevant conflicts of interest to declare.

Deregulation of the imprinted DLK1-DIO3 locus ncRNAs is associated with replicative senescence of human adipose-derived stem cells.

BackgroundHuman adult adipose-derived stem cells (hADSCs) have become the most promising cell source for regenerative medicine. However the prolonged ex vivo expansion periods required to obtain the necessary therapeutic dose promotes progressive senescence, with the concomitant reduction of their therapeutic potential.Aim and scopeA better understanding of the determinants of hADSC senescence is needed to improve biosafety while preserving therapeutic efficiency. Here, we investigated the association between deregulation of the imprinted DLK1-DIO3 region and replicative senescence in hADSC cultures.MethodsWe compared hADSC cultures at short (PS) and prolonged (PL) passages, both in standard and low [O2] (21 and 3%, respectively), in relation to replicative senescence. hADSCs were evaluated for expression alterations in the DLK1-DIO3 region on chromosome 14q32, and particularly in its main miRNA cluster.ResultsComparison of hADSCs cultured at PL or PS surprisingly showed a quite significant fraction (69%) of upregulated miRNAs in PL cultures mapping to the imprinted 14q32 locus, the largest miRNA cluster described in the genome. In agreement, expression of the lncRNA MEG3 (Maternally Expressed 3; Meg3/Gtl2), cultured at 21 and 3% [O2], was also significantly higher in PL than in PS passages. During hADSC replicative senescence the AcK16H4 activating mark was found to be significantly associated with the deregulation of the entire DLK1-DIO3 locus, with a secondary regulatory role for the methylation of DMR regions.ConclusionA direct relationship between DLK1-DIO3 deregulation and replicative senescence of hADSCs is reported, involving upregulation of a very significant fraction of its largest miRNA cluster (14q32.31), paralleled by the progressive overexpression of the lncRNA MEG3, which plays a central role in the regulation of Dlk1/Dio3 activation status in mice.

Relationship between Altered miRNA Expression and DNA Methylation of the DLK1-DIO3 Region in Azacitidine-Treated Patients with Myelodysplastic Syndromes and Acute Myeloid Leukemia with Myelodysplasia-Related Changes.

The DLK1–DIO3 region contains a large miRNA cluster, the overexpression of which has previously been associated with myelodysplastic syndromes (MDS). To reveal whether this overexpression is epigenetically regulated, we performed an integrative analysis of miRNA/mRNA expression and DNA methylation of the regulatory sequences in the region (promoter of the MEG3 gene) in CD34+ bone marrow cells from the patients with higher-risk MDS and acute myeloid leukemia with myelodysplasia-related changes (AML-MRC), before and during hypomethylating therapy with azacytidine (AZA). Before treatment, 50% of patients showed significant miRNA/mRNA overexpression in conjunction with a diagnosis of AML-MRC. Importantly, increased level of MEG3 was associated with poor outcome. After AZA treatment, the expression levels were reduced and were closer to those seen in the healthy controls. In half of the patients, we observed significant hypermethylation in a region preceding the MEG3 gene that negatively correlated with expression. Interestingly, this hypermethylation (when found before treatment) was associated with longer progression-free survival after therapy initiation. However, neither expression nor methylation status were associated with future responsiveness to AZA treatment. In conclusion, we correlated expression and methylation changes in the DLK1–DIO3 region, and we propose a complex model for regulation of this region in myelodysplasia.

The long noncoding RNA Bsr in the genomically imprinted Dlk1-Dio3 region is suppressed in newborn rat pancreas by gestational obesity

Abstract Background: Fetal metabolic programming imposed by maternal obesity and impaired glucose tolerance predisposes the offspring to metabolic disease and beta cell dysfunction as adults. The aim of this study was to assess the whole pancreas RNA changes in neonatal offspring exposed to fetal programming. Methods: The outcome of fetal programming on offspring was tested using the selectively bred Diet Induced Obese (DIO) and the Diet Resistant (DR) strains. DIO and DR rats were fed either chow or high fat, high sucrose (high energy, HE) diet during gestation and the differences in pancreas RNA expression at two days after birth were measured by microarray. Pancreas sections were stained and analyzed for alpha and beta cell numbers. Neonatal islets were treated with cytotoxic cytokines and RNA measured by RT-Q-PCR. Results: Morphometric analyses revealed significant differences in alpha and beta cell numbers per pancreas or per islet by HE diet. Microarray analyses revealed 11 fold downregulation of the long noncoding RNA Bsr in whole pancreas by HE feeding of DIO rat dams. MicroRNAs from the same locus, the Dlk1- Dio3 imprinted region, were also decreased by HE diet. Moreover, treatment of isolated neonatal islets with inflammatory cytokines, IL-1β and IFN-γ, led to reduction of Bsr transcript in a time and dose dependent manner. Conclusions: Our data suggest that fetal programming of Bsr may play a role in beta cell dysfunction in obesity and type 2 diabetes.

Abstract B67: The DLK1-DIO3 imprinted region regulates long-term proliferation in normal and malignant breast epithelium

Abstract DNA methylation changes are common in cancers, but their consequences are not fully understood. The DLK1-DIO3 locus, whose transcripts are controlled by the imprinted control region IG-DMR, is hypomethylated in 8% of breast cancer patients. Moreover, breast cancer patients with increased copy number of miR-127, an miR encoded in the DLK1-DIO3 locus, had worse disease-specific survival. IG-DMR reporter mice revealed that the elevated expression of miR-127 in immature normal and malignant mammary epithelial cells is controlled by allele-specific DNA methylation. Aberrant expression of miR-127 led to the de novo acquisition of long-term proliferation by normal and malignant mammary epithelial cells. Mechanistically, miR-127 suppresses translation of SETD8 and subsequent H4K20me1 deposition at LATS2, resulting in an effective activation of the Hippo target genes. Our results demonstrate that the DLK1-DIO3 region epigenetically regulates mammary stem cells, contributing to increased proliferative and tumorigenic capacity partially via regulation of the Hippo signaling pathway. Citation Format: Maider Zabala, Neethan A. Lobo, Jose A. Seoane, Yonatan Stelzer, An V. Luong, Taichi Isobe, Mark A. Zarnegar, Nicholas Watanabe, Sonia Antoñana, Jessica Lam, Dalong Qian, Shaheen S. Sikandar, Angera H. Kuo, Luuk S. Heitink, Yohei Shimono, Ferenc A. Scheeren, Shang Cai, Shigeo Hisamori, Debashis Sahoo, Frederick M. Dirbas, George Somlo, Rudolf Jaenisch, Curtis Christina, Michael F. Clarke. The DLK1-DIO3 imprinted region regulates long-term proliferation in normal and malignant breast epithelium [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B67.

JAK/STAT3 regulated global gene expression dynamics during late-stage reprogramming process

BackgroundThe generation of induced pluripotent stem cells (iPSCs) has underdefined mechanisms. In addition, leukemia inhibitory factor (LIF) activated Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway is the master regulator for naïve-state pluripotency achievement and maintenance. However, the regulatory process to attain naïve pluripotent iPSCs is not well understood.ResultsWe performed transcriptome analysis to dissect the genomic expression during mouse iPSC induction, with or without blocking the JAK/STAT3 activity. We describe JAK/STAT3 signaling-specific biological events such as gametogenesis, meiotic/mitotic cell cycle, and DNA repair, and JAK/STAT3-dependent expression of key transcription factors such as the naïve pluripotency-specific genes, developmental pluripotency associated (Dppa) family, along with histone modifiers and non-coding RNAs in reprogramming. We discover that JAK/STAT3 activity does not affect early phase mesenchymal to epithelial transition (MET) but is necessary for proper imprinting of the Dlk1-Dio3 region, an essential event for pluripotency achievement at late-reprogramming stage. This correlates with the JAK/STAT3-dependent stimulation of Dppa3 and Polycomb repressive complex 2 (PRC2) genes. We further demonstrate that JAK/STAT3 activity is essential for DNA demethylation of pluripotent loci including Oct4, Nanog, and the Dlk1-Dio3 regions. These findings correlate well with the previously identified STAT3 direct targets. We further propose a model of pluripotency achievement regulated by JAK/STAT3 signaling during the reprogramming process.ConclusionsOur study illustrates novel insights for JAK/STAT3 promoted pluripotency establishment, which are valuable for further improving the naïve-pluripotent iPSC generation across different species including humans.

The Activity-Induced Long Non-Coding RNA Meg3 Modulates AMPA Receptor Surface Expression in Primary Cortical Neurons.

Transcription of new RNA is crucial for maintaining synaptic plasticity, learning and memory. Although the importance of synaptic plasticity-related messenger RNAs (mRNAs) is well established, the role of a large group of long non-coding RNAs (lncRNAs) in long-term potentiation (LTP) is not known. In this study, we demonstrated the expression of a lncRNA cluster, namely maternally expressed gene 3 (Meg3), retrotransposon-like gene 1-anti-sense (Rtl1-AS), Meg8 and Meg9, which is located in the maternally imprinted Dlk1-Dio3 region on mouse chromosome 12qF1, in primary cortical neurons following glycine stimulation in an N-Methyl-D-aspartate receptor (NMDAR)-dependent manner. Importantly, we also validated the expression of Meg3, Meg8 and Meg9 in the hippocampus of mice following cued fear conditioning in vivo. Interestingly, Meg3 is the only lncRNA that is expressed in the nucleus and cytoplasm. Further analysis revealed that Meg3 loss of function blocked the glycine-induced increase of the GluA1 subunit of AMPA receptors on the plasma membrane, a major hallmark of LTP. This aberrant trafficking of AMPA receptors correlated with the dysregulation of the phosphatidylinoside-3-kinase (PI3K)/AKT signaling pathway and the downregulation of the lipid phosphatase and tensin homolog (PTEN). These findings provide the first evidence for a functional role of the lncRNA Meg3 in the intricate regulation of the PTEN/PI3K/AKT signaling cascade during synaptic plasticity in neurons.

High throughput sequencing identifies an imprinted gene, Grb10, associated with the pluripotency state in nuclear transfer embryonic stem cells.

Somatic cell nuclear transfer and transcription factor mediated reprogramming are two widely used techniques for somatic cell reprogramming. Both fully reprogrammed nuclear transfer embryonic stem cells and induced pluripotent stem cells hold potential for regenerative medicine, and evaluation of the stem cell pluripotency state is crucial for these applications. Previous reports have shown that the Dlk1-Dio3 region is associated with pluripotency in induced pluripotent stem cells and the incomplete somatic cell reprogramming causes abnormally elevated levels of genomic 5-methylcytosine in induced pluripotent stem cells compared to nuclear transfer embryonic stem cells and embryonic stem cells. In this study, we compared pluripotency associated genes Rian and Gtl2 in the Dlk1-Dio3 region in exactly syngeneic nuclear transfer embryonic stem cells and induced pluripotent stem cells with same genomic insertion. We also assessed 5-methylcytosine and 5-hydroxymethylcytosine levels and performed high-throughput sequencing in these cells. Our results showed that Rian and Gtl2 in the Dlk1-Dio3 region related to pluripotency in induced pluripotent stem cells did not correlate with the genes in nuclear transfer embryonic stem cells, and no significant difference in 5-methylcytosine and 5-hydroxymethylcytosine levels were observed between fully and partially reprogrammed nuclear transfer embryonic stem cells and induced pluripotent stem cells. Through syngeneic comparison, our study identifies for the first time that Grb10 is associated with the pluripotency state in nuclear transfer embryonic stem cells.

Abstract 107: Mir-431 as a Potential Master Regulator in Angiotensin Ii-induced Vascular Injury

Objective: Vascular injury is an early manifestation and a cause of end-organ damage in hypertension. microRNAs (miRNAs) play an important role in cardiovascular disease, but their implication in vascular injury is remains unclear. We aim to use RNA sequencing (seq) and a systems biology approach to identify master regulators that mediate global gene expression changes in the course of vascular injury. Methods and Results: Ten week-old male C57BL/6 mice were infused or not with angiotensin (Ang) II (1 μg/kg/min, SC) for 14 days. Blood pressure (BP) was measured by telemetry. Total RNA was extracted from the mesenteric vasculature for total RNA and small RNA-seq. Differentially expressed (DE) miRNAs (23 up and 12 down) and mRNAs (550 up and 256 down) were identified (1.5-fold, q <0.05). Molecular networks were constructed to integrate predicted interactions between DE miRNAs and inversely expressed DE mRNAs and between DE transcription factors (TF) and DE genes. Gene enrichment analysis revealed DE mRNAs involved in extracellular matrix (ECM) and developmental processes regulated by DE miRNAs ( q <1.5E-11). Seventeen upregulated miRNAs are located in the miRNA cluster of the Dlk1-Dio3 region that is highly conserved in humans, 9 of which had expression levels correlated with BP ( P <0.05). Among those 9, miR-431 that ranked first as DE miRNA ( q <0.0005) and is 100% conserved in humans, and a conserved putative DE target, a BP-correlated ( P <0.05) TF ETS homologous factor ( Ehf ), which regulates numerous ECM genes including collagen type I α1 ( Col1a1 ), were selected for functional studies. Transfection of a miR-431 mimic in human aortic smooth muscle cells (HASMCs) decreased Ehf (0.1±0.1-fold, P <0.001) and increased Ehf -suppressing target Col1a1 (1.7±0.5-fold, P <0.001) mRNA levels. Transfection of a miR-431 inhibitor caused reciprocal effects ( P <0.05). Ehf siRNA knockdown increased Col1a1 (1.2±0.1-fold, P <0.001) mRNA levels. Conclusions: Ang II infusion altered expression of miRNAs in the Dlk1-Dio3 cluster and genes involved in ECM and developmental processes. miR-431 targets TF Ehf , which leads to increased Col1a1 in HASMCs. miR-431 may act as a master regulator for vascular injury and could be a potential therapeutic target.

The Value of miR-299-5p in Diagnosis and Prognosis of Intestinal-Type Gastric Adenocarcinoma.

MicroRNAs (miRNAs) are a class of non-coding RNAs, containing about 22 nucleotides and having a pivotal function in various cellular processes. The oncogenic and tumor suppressor roles of miRNAs have been identified in cancers especially in gastric cancer, which is one of the most prevalent cancers. MiR-299-5p is located in the imprinted Dlk1-Dio3 region in chromosome 14q32. Aberrant expression of miR-299-5p was determined in solid and blood cancers. The current study was performed to assess the expression pattern of miR-299-5p in intestinal-type gastric adenocarcinoma and compare it with the normal adjacent counterparts. The expression level of miR-299-5p was investigated in forty fresh specimens which were obtained from gastric cancer patients during endoscopy. Moreover, the association of aberrant expression of miR-299-5p and clinicopathological features, as well as the susceptibility of miR-299-5p as a tumor marker, was determined. The result of qRT-PCR revealed the downregulation of miR-299-5p in intestinal-type gastric adenocarcinoma compared with adjacent tumor-free tissues (P<0.001); this misregulation can be used as a tumor marker. Analysis of miR-299-5p misregulation did not reveal a significant correlation with clinical features. The result obtained from the present study revealed the significant downregulation of miR-299-5p in intestinal-type gastric adenocarcinoma which is consistent with previous studies showing miR-299-5p downregulation in other types of cancers. The data obtained from the current study suggest basic information which can be very helpful for future research in the field of diagnosis and treatment of gastric cancer.

Abstract B08: Expression of noncoding RNAs on the DLK1-DIO1 locus in EMT and breast cancer

Abstract The DLK1-DIO3 locus contains three paternally expressed protein-coding genes and maternally expressed non-protein coding genes, including the lncRNA MEG3 and a cluster of 54 miRNA, making it one of the largest miRNA clusters in the genome. A recent study showed that the degree of activation of the maternally inherited homolog of this region was correlated with the pluripotency of stem cells. Also, the maternal ncRNAs from the DLK1-DIO3 locus are amongst the few regions in the transcriptome able to discriminate between induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), thereby linking this locus to the maintenance of stem cell character. In humans DLK1-DIO3-derived ncRNAs have been linked to both progression and inhibition of cancer. The objective of this study was to analyze the expression profile of the DLK1-DIO3-derived ncRNAs and in particular the lncRNA MEG3 in breast epithelial progenitor cell lines (D492 and HMLE) with high degree of plasticity towards epithelial to mesenchymal transition (EMT) and cancer progression. D492M and HMLEmes are mesenchymal derivatives of D492 and HMLE, respectively. Total RNA and small RNA sequencing revealed marked upregulation of the maternally expressed lncRNAs and the miRNAs on the DLK1-DIO3 locus in D492M compared to D492. This miRNA-cluster along with the lncRNA MEG3 is expressed from the maternal inherited homolog. Allele specific expression analysis shows that the increased expression of MEG3 in D492M is not caused by loss of imprinting but increased expression of the already expressed allele. These ncRNAs are also upregulated in HMLEmes compared to HMLE, showing that these effects are not cell line specific. qPCR analysis of primary tissue shows that MEG3 expression is 8-15 fold higher in stromal cells than epithelial cells of the breast. These results indicate that the MEG3 upregulation and/or the maternally expressed miRNA cluster could be critical regulators/effectors in the EMT/mesenchymal phenotype. Data from the The Cancer Genome Atlas consistently shows positive correlation between the expression of Meg3 and the miRNAs located at the DLK1-DIO3 genomic region. We have characterized the main splice-forms of the MEG3 gene, expressed in D492 and D492M. Out of the 12 different splice-forms, splice-form 1 (NR_002766) was mostly responsible for increased expression in D492M. Since EMT has been linked to metastatic behavior of epithelial-originating cancers we measured the expression of MEG3 in a series of clinically well-defined breast tumors (N= 125). Survival analysis showed lower overall survival in patients with high MEG3 expression (P=0.01). Comparison with expression and survival data from the online GOBO (Gene Expression-Based Outcome for Breast Cancer Online) database confirmed our observed negative relationship between MEG3 expression and survival which was particularly evident in luminal B type tumors. MEG3 is also negatively correlated with distant metastasis-free survival of poorly differentiated (grade 3) tumors, suggesting that MEG3 expression may be a valid marker of EMT during tumorigenesis. Conclusions: ncRNAs are increasingly seen as intrinsic regulators of stemness, tissue morphogenesis and cancer progression. Our data indicates that the maternally expressed ncRNAs at the DLK1-DIO3 region could be regulators of EMT in breast cancer. Future works will aim at understanding the biological role of MEG3 and the miRNA-cluster in these processes. Citation Format: Bylgja Hilmarsdottir, Eirikur Briem, Jon Thor Bergthorsson, Thorarinn Gudjonsson, Magnus Karl Magnusson. Expression of noncoding RNAs on the DLK1-DIO1 locus in EMT and breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B08.

MiR-377-dependent BCL-xL regulation drives chemotherapeutic resistance in B-cell lymphoid malignancies.

BackgroundBCL-xL is an anti-apoptotic BCL-2 family protein that inhibits apoptosis and is overexpressed in many cancers. We have reported that acquired resistance to the BCL-2 inhibitor ABT-199 (venetoclax) is associated with increased BCL-xL expression. Yet, how BCL-xL mediates chemoresistance in hematopoietic malignancies is not clear. This finding may help in design of new strategies for therapeutic intervention to overcome acquired chemoresistance mediated by BCL-xL.ResultsWe now show that the increased BCL-xL expression was inversely correlated with that of miR-377 in ABT-199-resistant cells. This finding was also extended to a panel of B-cell lymphoid lines and primary chronic lymphocytic leukemia (CLL) cells. miR-377 suppressed BCL-xL expression by recognizing two binding sites in the BCL-xL 3’-UTR. Mutation of these two miR-377 consensus-binding sites completely abolished its regulatory effect. Expression of a miR-377 mimic downregulated BCL-xL protein expression and significantly increased apoptotic cell death. Expression of a miR-377 inhibitor restored BCL-xL protein expression and limited cell death caused by the hypomethylating agent 5-azacytidine. Thus, miR-377-dependent BCL-xL regulation drives acquired therapeutic resistance to ABT-199. We further show that CLL patients who received a diverse array of chemotherapy regimens also had significantly higher BCL-xL and lower miR377 expression, indicating that exposure to chemotherapy might trigger transcriptional silencing of miR-377, which results in high levels of BCL-xL. Importantly, CLL patients with high BCL-xL/low miR-377 expression had an advanced tumor stage. Moreover, the high BCL-xL expression correlated with short treatment-free survival in 76 CLL patients. miR-377 is located at 14q32 in the DLK1-DIO3 region, which encodes the largest tumor suppressor miRNA cluster in humans. Examination of five additional 14q32 miRNAs revealed that the majority were significantly down-regulated in most CLL patients as well as in ABT-199-resistant cell lines. Remarkably, four of these miRNAs had significantly decreased expression in chemotherapy-treated CLL patients as compared to those untreated. These findings indicate a reduced expression of multiple miRNAs that may reflect a global silencing of this miRNA cluster in therapy-resistant lymphoid cells.ConclusionsThese findings reveal a novel mechanism by which down-regulation of miR-377 increases BCL-xL expression, promoting chemotherapy resistance in B-cell lymphoid malignancies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0460-8) contains supplementary material, which is available to authorized users.

Ascorbic acid improves pluripotency of human parthenogenetic embryonic stem cells through modifying imprinted gene expression in the Dlk1-Dio3 region.

IntroductionHuman parthenogenetic embryonic stem cells (hpESCs) are generated from artificially activated oocytes, however, the issue of whether hpESCs have equivalent differentiation ability to human fertilized embryonic stem cells remains controversial.MethodshpESCs were injected into male severe combined immunodeficiency (SCID) mice and the efficiency of teratoma formation was calculated. Then the gene expression and methylation modification were detected by real time-PCR and bisulfate methods.ResultsComparison of five hpESCs with different differentiation abilities revealed that levels of paternal genes in the Dlk1-Dio3 region on chromosome 14 in the hpESCs with high differentiation potential are enhanced, but strictly methylated and silenced in the hpESCs with lower differentiation potential. Treatment with ascorbic acid, rescued their ability to support teratoma formation and altered the expression profiles of paternally expressed genes in hpESCs that could not form teratoma easily. No differences in the expression of other imprinting genes were evident between hpESCs with higher and lower differentiation potential, except for those in the Dlk1-Dio3 region.ConclusionsThe Dlk1-Dio3 imprinting gene cluster distinguishes the differentiation ability of hpESCs. Moreover, modification by ascorbic acid may facilitate application of hpESCs to clinical settings in the future by enhancing their pluripotency.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0054-9) contains supplementary material, which is available to authorized users.