MicroRNAs in the Imprinted DLK1-DIO3 Region Repress the Epithelial-to-Mesenchymal Transition by Targeting the TWIST1 Protein Signaling Network

Development of metastatic disease accounts for the vast majority of cancer-related deaths. Nevertheless, few treatments exist that are designed to specifically inhibit processes that drive tumor metastasis. The imprinted DLK1-DIO3 region contains tumor-suppressing miRNAs, but their identity and function remain indeterminate. In this study we identify seven miRNAs in the imprinted DLK1-DIO3 region that function cooperatively to repress the epithelial-to-mesenchymal transition, a critical step that drives tumor metastasis, as well as proliferation of carcinoma cells. These seven miRNAs (miRs 300, 382, 494, 495, 539, 543, and 544) repress a signaling network comprising TWIST1, BMI1, ZEB1/2, and miR-200 family miRNAs and silencing of the cluster, which occurs via hypermethylation of upstream CpG islands in human ductal carcinomas, confers morphological, molecular, and function changes consistent with an epithelial-to-mesenchymal transition. Moreover, ectopic expression of miR-544 independently inhibited proliferation of numerous tumor cell lines by inducing the ATM cell cycle checkpoint pathway. These results establish the DLKI-DIO3 miRNA cluster as a critical checkpoint regulating tumor growth and metastasis and implicate epigenetic modification of the cluster in driving tumor progression. These results also suggest that promoter methylation status and miRNA expression levels represent new diagnostic tools and therapeutic targets to predict and inhibit, respectively, tumor metastasis in carcinoma patients.     

哺乳动物印记域DLK1-DIO3的研究进展

DLK1-DIO3印记域定位于人14号染色体、小鼠12号染色体及绵羊18号染色体远端, 在真哺乳亚纲动物中印记保守。该印记域包含3个编码蛋白的父系表达基因Dlk1、Rtl1和Dio3以及若干大小不同的母系表达印记非编码RNA, 如miRNAs、snoRNAs 和大型非编码RNA Gtl2等。人和小鼠该印记域内印记基因剂量的改变将导致严重的表型异常甚至胚胎致死, 暗示正常的发育需要域内印记基因的正常表达。文章重点论述了哺乳动物DLK1-DIO3印记域的印记调控机制和域内印记基因及其功能的研究进展。     

DLK1-DIO3印记区域的miRNAs与疾病的发生

哺乳动物的基因组以发育调控模式进行转录,生成长的和短的非编码RNAs(non-coding RNA,ncRNAs).ncRNAs占到人类转录组的98%,与生物体进化复杂程度显著相关.MicroRNAs(miRNAs)是目前研究比较透彻的,长度大约为20~24个核苷酸的ncRNAs,其通过与靶基因mRNA的结合在转录后水平负调控基因的表达.人类基因组中一个最大的miRNA簇位于14号染色体(14q32)的DLK1-DIO3印记区域,包括了54个miRNAs.这些miRNAs通过参与调节重要的信号通路在许多病理过程中发挥作用.充分了解DLK1-DIO3印记区域中这个大的miRNA簇,在病理生理过程中的重要性将有助于为相关疾病的治疗提供新的策略.本文比较深入地分析了DLK1-DIO3印记区域中的miRNAs在调控组织动态平衡以及多种癌症发生中的作用,同时对其潜在的临床应用价值进行了讨论.     

Molecular mechanisms regulating phenotypic outcome in paternal and maternal uniparental disomy for chromosome 14

Human chromosome 14q32.2 carries a cluster of imprinted genes including paternally expressed genes (PEGs) such as DLK1 and RTL1, and maternally expressed genes (MEGs) such as GTL2 (alias, MEG3), RTL1as (RTL1 antisense), and MEG8. Consistent with this, paternal and maternal uniparental disomies for chromosome 14 (upd(14)pat and upd(14)mat) cause distinct phenotypes. In this report, we review the current knowledge about the underlying factors for the development of clinical features in upd(14)pat and upd(14)mat. The data available suggest that the DLK1–GTL2 IG-DMR functions as a regulator for the maternally inherited imprinted region, and that excessive RTL1 expression and decreased DLK1 and RTL1 expression play a major role in the development of upd(14)pat-like and upd(14)mat-like phenotypes, respectively     

The Upregulation of Genomic Imprinted DLK1-Dio3 miRNAs in Murine Lupus Is Associated with Global DNA Hypomethylation

Epigenetic factors such as DNA methylation and microRNAs (miRNAs) are now increasingly recognized as vital contributors to lupus etiology. In this study, we investigated the potential interaction of these two epigenetic factors in lupus-prone MRL-lpr mice. We recently reported dysregulated expression of miRNAs in splenocytes of MRL-lpr mice. Here, we report that a majority of the upregulated miRNAs in MRL-lpr mice is located at the genomic imprinted DLK1-Dio3 domain. Further, we show a differential magnitude of upregulation of DLK1-Dio3 miRNA cluster in purified splenic CD4⁺ T, CD19⁺ B, and splenic CD4^-CD19^- cells from MRL-lpr lupus mice when compared to control MRL mice. MRL-lpr splenocytes (especially CD19⁺ and CD4^-CD19^- subsets) were hypomethylated compared to cells from control, MRL mice. We further show that deliberate demethylation of splenocytes from control MRL mice, but not from MRL-lpr lupus mice, with specific DNA methylation inhibitor 5-Aza-2’-deoxycytidine significantly augmented DLK1-Dio3 miRNAs expression. These findings strongly indicate that the upregulation of DLK1-Dio3 miRNAs in lupus splenic cell subsets is associated with reduced global DNA methylation levels in lupus cells. There was a differential upregulation of DLK-Dio3 miRNAs among various demethylated splenic cell subsets, which implies varied sensitivity of DLK1-Dio3 miRNA cluster in these cell subsets to DNA hypomethylation. Finally, inhibition of select DLK1-Dio3 miRNA such as miR-154, miR-379 and miR-300 with specific antagomirs significantly reduced the production of lupus-relevant IFNγ, IL-1β, IL-6, and IL-10 in lipopolysaccharide (LPS) activated splenocytes from MRL-lpr mice. Our study is the first to show that DNA methylation regulates genomic imprinted DLK1-Dio3 miRNAs in autoimmune lupus, which suggests a connection of DNA methylation, miRNA and genomic imprinting in lupus pathogenesis.     

Methylation analysis of the imprinted DLK1-GTL2 domain supports the random parental origin of the IGH-involving del(14q) in B-cell malignancies

Leukemias/lymphomas with IGH-involving del(14q)¹ commonly lose the DLK1-GTL2 imprinted domain that comprises several paternally and maternally expressed genes, including a cluster of microRNAs. Given that deletion of this region could lead to inactivation of a monoallelically expressed tumor suppressor gene, our study aimed at determination of the parental origin of del(14q/IGH). The designed allele-specific methylation study of the DLK1/GTL2 intergenic differentially methylated region allowed us to determine the parental origin of del(14q/IGH) in 9/20 analyzed cases. In six cases del(14q/IGH) was of the paternal origin and in three cases of the maternal origin. These findings argue against the concept that a TSG/anti-oncomir located in the imprinted region is systematically inactivated by a targeted deletion of its functional allele.     

The IG-DMR and the MEG3-DMR at Human Chromosome 14q32.2: Hierarchical Interaction and Distinct Functional Properties as Imprinting Control Centers

Human chromosome 14q32.2 harbors the germline-derived primary DLK1-MEG3 intergenic differentially methylated region (IG-DMR) and the postfertilization-derived secondary MEG3-DMR, together with multiple imprinted genes. Although previous studies in cases with microdeletions and epimutations affecting both DMRs and paternal/maternal uniparental disomy 14-like phenotypes argue for a critical regulatory function of the two DMRs for the 14q32.2 imprinted region, the precise role of the individual DMR remains to be clarified. We studied an infant with upd(14)pat body and placental phenotypes and a heterozygous microdeletion involving the IG-DMR alone (patient 1) and a neonate with upd(14)pat body, but no placental phenotype and a heterozygous microdeletion involving the MEG3-DMR alone (patient 2). The results generated from the analysis of these two patients imply that the IG-DMR and the MEG3-DMR function as imprinting control centers in the placenta and the body, respectively, with a hierarchical interaction for the methylation pattern in the body governed by the IG-DMR. To our knowledge, this is the first study demonstrating an essential long-range imprinting regulatory function for the secondary DMR.     

Over-expression of the miRNA cluster at chromosome 14q32 in the alcoholic brain correlates with suppression of predicted target mRNA required for oligodendrocyte proliferation

We examined miRNA expression from RNA isolated from the frontal cortex (Broadman area 9) of 9 alcoholics (6 males, 3 females, mean age 48 years) and 9 matched controls using both the Affymetrix GeneChip miRNA 2.0 and Human Exon 1.0 ST Arrays to further characterize genetic influences in alcoholism and the effects of alcohol consumption on predicted target mRNA expression. A total of 12 human miRNAs were significantly up-regulated in alcohol dependent subjects (fold change ≥ 1.5, false discovery rate (FDR) ≤ 0.3; p < 0.05) compared with controls including a cluster of 4 miRNAs (e.g., miR-377, miR-379) from the maternally expressed 14q32 chromosome region. The status of the up-regulated miRNAs was supported using the high-throughput method of exon microarrays showing decreased predicted mRNA gene target expression as anticipated from the same RNA aliquot. Predicted mRNA targets were involved in cellular adhesion (e.g., THBS2), tissue differentiation (e.g., CHN2), neuronal migration (e.g., NDE1), myelination (e.g., UGT8, CNP) and oligodendrocyte proliferation (e.g., ENPP2, SEMA4D1). Our data support an association of alcoholism with up-regulation of a cluster of miRNAs located in the genomic imprinted domain on chromosome 14q32 with their predicted gene targets involved with oligodendrocyte growth, differentiation and signaling.     

Epigenetic regulation of microRNAs in cancer: an integrated review of literature

MicroRNAs (miRNAs) belong to the heterogeneous class of non-coding RNAs (ncRNAs) that regulate the translation and degradation of target mRNAs, and control approximately 30% of human genes. MiRNA genes might be silenced in human tumors (oncomiRs) by aberrant hypermethylation of CpG islands that encompass or lie adjacent to miRNA genes and/or by histone modifications. We performed literature search for research articles describing epigenetically regulated miRNAs in cancer and identified 45 studies that were published between 2006 and 7/2010. The data from those papers are fragmented and methodologically heterogeneous and our work represents first systematic review towards to integration of diverse sets of information. We reviewed the methods used for detection of miRNA epigenetic regulation, which comprise bisulfite genomic sequencing PCR (BSP), bisulfite pyrosequencing, methylation specific PCR (MSP), combined bisulfite restriction analysis (COBRA), methylation sensitive single nucleotide primer extension (Ms-SNuPE), MassARRAY technique and some modifications of those methods. This integrative study revealed 122 miRNAs that were reported to be epigenetically regulated in 23 cancer types. Compared to protein coding genes, human oncomiRs showed an order of magnitude higher methylation frequency (11.6%; 122/1048 known miRNAs). Nearly half, (45%; 55/122) epigenetically regulated miRNAs were associated with different cancer types, but other 55% (67/122) miRNAs were present in only one cancer type and therefore representing cancer-specific biomarker potential. The data integration revealed miRNA epigenomic hot spots on the chromosomes 1q, 7q, 11q, 14q and 19q. CpG island analysis of corresponding miRNA precursors (pre-miRNAs) revealed that 20% (26/133) of epigenetically regulated miRNAs had a CpG island within the range of 5kb upstream, among them 14% (19/133) of miRNAs resided within the CpG island. Our integrative survey and analyses revealed candidate cancer-specific miRNA epigenetic signatures which provide the basis for new therapeutic strategies in cancer by targeting the epigenetic regulation of miRNAs.     

Paternal uniparental disomy 14 and related disorders: Placental gene expression analyses and histological examinations

Although recent studies in patients with paternal uniparental disomy 14 [upd(14)pat] and other conditions affecting the chromosome 14q32.2 imprinted region have successfully identified underlying epigenetic factors involved in the development of upd(14)pat phenotype, several matters, including regulatory mechanism(s) for RTL1 expression, imprinting status of DIO3 and placental histological characteristics, remain to be elucidated. We therefore performed molecular studies using fresh placental samples from two patients with upd(14)pat. We observed that RTL1 expression level was about five times higher in the placental samples of the two patients than in control placental samples, whereas DIO3 expression level was similar between the placental samples of the two patients and the control placental samples. We next performed histological studies using the above fresh placental samples and formalin-fixed and paraffin-embedded placental samples obtained from a patient with a maternally derived microdeletion involving DLK1, the-IG-DMR, the MEG3-DMR and MEG3. Terminal villi were associated with swollen vascular endothelial cells and hypertrophic pericytes, together with narrowed capillary lumens. DLK1, RTL1 and DIO3 proteins were specifically identified in vascular endothelial cells and pericytes, and the degree of protein staining was well correlated with the expression dosage of corresponding genes. These results suggest that RTL1as-encoded microRNA functions as a repressor of RTL1 expression, and argue against DIO3 being a paternally expressed gene. Furthermore, it is inferred that DLK1, DIO3 and, specially, RTL1 proteins, play a pivotal role in the development of vascular endothelial cells and pericytes.     

Paternal uniparental disomy 14 and related disorders

Although recent studies in patients with paternal uniparental disomy 14 [upd(14)pat] and other conditions affecting the chromosome 14q32.2 imprinted region have successfully identified underlying epigenetic factors involved in the development of upd(14)pat phenotype, several matters, including regulatory mechanism(s) for RTL1 expression, imprinting status of DIO3 and placental histological characteristics, remain to be elucidated. We therefore performed molecular studies using fresh placental samples from two patients with upd(14)pat. We observed that RTL1 expression level was about five times higher in the placental samples of the two patients than in control placental samples, whereas DIO3 expression level was similar between the placental samples of the two patients and the control placental samples. We next performed histological studies using the above fresh placental samples and formalin-fixed and paraffin-embedded placental samples obtained from a patient with a maternally derived microdeletion involving DLK1, the-IG-DMR, the MEG3-DMR and MEG3. Terminal villi were associated with swollen vascular endothelial cells and hypertrophic pericytes, together with narrowed capillary lumens. DLK1, RTL1 and DIO3 proteins were specifically identified in vascular endothelial cells and pericytes, and the degree of protein staining was well correlated with the expression dosage of corresponding genes. These results suggest that RTL1as-encoded microRNA functions as a repressor of RTL1 expression, and argue against DIO3 being a paternally expressed gene. Furthermore, it is inferred that DLK1, DIO3 and, specially, RTL1 proteins, play a pivotal role in the development of vascular endothelial cells and pericytes.     

Global regulation on microRNA in hepatitis B virus-associated hepatocellular carcinoma

Recent work has revealed the causative links between deregulation of microRNAs (miRNAs) and cancer development. In hepatocellular carcinoma (HCC), aberrant expression of miRNAs has been observed, but the molecular mechanisms that contribute to such changes remains to be elucidated. Here, we reported the analysis of miRNA expression in 94 pairs of tumor and adjacent nontumor tissues from HBV-associated HCC in Chinese patients. We found miRNAs were aberrantly expressed in HCC tissues. To investigate the cause of such deregulation, we detected changes in DNA copy number by measuring locus-specific hybridization intensity, and found changes in expression of several miRNAs are correlated with genomic amplification or deletion. For example, the genomic regions of miR-30d and miR-151 were amplified in ～50% of HCC tumor tissues, and the expressions of these miRNAs are significantly correlated with DNA copy number. We also employed cDNA microarray data, and provide evidence that key regulators of the miRNA biosynthetic pathway, including DROSHA, DGCR8, AGO1, and AGO2, are frequently overexpressed in HCC. This study provides molecular clues that may contribute to the global changes of miRNA expression in HCC.     

miR-150-5p Inhibits Hepatoma Cell Migration and Invasion by Targeting MMP14

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality worldwide. Despite progress in diagnostics and treatment of HCC, its prognosis remains poor because the molecular mechanisms underlying hepatocarcinogenesis are not well understood. In the study, we focused on identifying the role of miRNAs in HCC progression. miRNA microarray was used to analyze the differentially expressed miRNAs, and the results were validated by qPCR. We found that the miR-150-5p expression is down-regulated in HCC tissues compared with pair non-tumor tissues. miR-150-5p expression is also decreased in metastatic cancer tissues compared with pair primary tissues, indicating that miR-150-5p may be involved in HCC metastasis. Functionally, miR-150-5p inhibition significantly promotes hepatoma cell migration and invasion, whereas miR-150-5p overexpression suppresses cancer cell migration and invasion in vitro. The matrix metalloproteinase 14 (MMP14) is identified as a new target gene of miR-150-5p. miR-150-5p markedly inhibits MMP14 expression in hepatoma cells, and miR-150-5p expression is negative correlation with MMP14 expression in vivo. More important, re-expression of MMP14 in hepatoma cells partially reverses the effect of miR-150-5p in inhibiting cell invasion.