Branching epithelial morphogenesis is closely linked to epithelial-to-mesenchymal transition (EMT), a process important in normal development and cancer progression. The miR-200 family regulates ...epithelial morphogenesis and EMT through a negative feedback loop with the ZEB1 and ZEB2 transcription factors. miR-200 inhibits expression of ZEB1/2 mRNA, which in turn can down-regulate the miR-200 family that further results in down-regulation of E-cadherin and induction of a mesenchymal phenotype. Recent studies show that the expression of miR-200 genes is high during late pregnancy and lactation, thereby indicating that these miRs are important for breast epithelial morphogenesis and differentiation. miR-200 genes have been studied intensively in relation to breast cancer progression and metastasis, where it has been shown that miR-200 members are down-regulated in basal-like breast cancer where the EMT phenotype is prominent. There is growing evidence that the miR-200 family is up-regulated in distal breast metastasis indicating that these miRs are important for colonization of metastatic breast cancer cells through induction of mesenchymal to epithelial transition. The dual role of miR-200 in primary and metastatic breast cancer is of interest for future therapeutic interventions, making it important to understand its role and interacting partners in more detail.
Epithelial-to-mesenchymal transition (EMT) and its reversed process mesenchymal-to-epithelial transition (MET) play a critical role in epithelial plasticity during development and cancer progression. ...Among important regulators of these cellular processes are non-coding RNAs (ncRNAs). The imprinted DLK1-DIO3 locus, containing numerous maternally expressed ncRNAs including the lncRNA maternally expressed gene 3 (
MEG3
) and a cluster of over 50 miRNAs, has been shown to be a modulator of stemness in embryonic stem cells and in cancer progression, potentially through the tumor suppressor role of
MEG3
. In this study we analyzed the expression pattern and functional role of ncRNAs from the DLK1-DIO3 locus in epithelial plasticity of the breast. We studied their expression in various cell types of breast tissue and revisit the role of the locus in EMT/MET using a breast epithelial progenitor cell line (D492) and its isogenic mesenchymal derivative (D492M). Marked upregulation of ncRNAs from the DLK1-DIO3 locus was seen after EMT induction in two cell line models of EMT. In addition, the expression of
MEG3
and the maternally expressed ncRNAs was higher in stromal cells compared to epithelial cell types in primary breast tissue. We also show that expression of
MEG3
is concomitant with the expression of the ncRNAs from the DLK1-DIO3 locus and its expression is therefore likely indicative of activation of all ncRNAs at the locus.
MEG3
expression is correlated with stromal markers in normal tissue and breast cancer tissue and negatively correlated with the survival of breast cancer patients in two different cohorts. Overexpression of
MEG3
using CRISPR activation in a breast epithelial cell line induced partial EMT and enriched for a basal-like phenotype. Conversely, knock down of
MEG3
using CRISPR inhibition in a mesenchymal cell line reduced the mesenchymal and basal-like phenotype of the cell line. In summary our study shows that maternally expressed ncRNAs are markers of EMT and suggests that
MEG3
is a novel regulator of EMT/MET in breast tissue. Nevertheless, further studies are needed to fully dissect the molecular pathways influenced by non-coding RNAs at the DLK1-DIO3 locus in breast tissue.
Objectives
To estimate the frequency of iron deficiency (ID) and anaemia in blood donors in Iceland and the impact of serum ferritin (SF) testing policy change.
Background
Blood donations contribute ...to ID and/or anaemia in whole blood donors (WBD). SF may be used to monitor blood donor iron stores.
Materials and Methods
The study included WBD and new donors (ND) in the Icelandic Blood Bank in 1997–2019. SF was measured for ND and intermittently for WBD until October 2017, but thereafter for all WBD and ND at every visit. In January 2018, the SF threshold increased from 14 to 16 μg/L for ND and from 8 to 10 μg/L for WBD.
Results
The study included 85 370 SF results from 243 369 visits of 32 910 donors. Median SF was higher for males than females, both for ND (88.0 vs. 31.2 μg/L, p < 0.001) and WBD (before 2018: 43.0 vs. 22.0 μg/L, p < 0.001). After the policy change in 2018, median SF increased for both male WBD (to 45.2 μg/L, p < 0.001) and female WBD (to 25.7 μg/L, p < 0.001). ID (SF <15 μg/L) was present in 10.6% of female ND and 0.5% of male ND. After policy change, the proportion of WB donations associated with ID decreased for males (from 6.4% to 4.0%) and females (from 18.9% to 14.1%). ID anaemia was present at some time in 3.7% of female WBD and 1.2% of male WBD.
Conclusion
This nationwide study showed that ID in WB donors is common, especially among females, but monitoring SF may improve donor management.
Gene expression signatures from microarray experiments promise to provide important prognostic tools for predicting disease outcome or response to treatment. A number of microarray studies in various ...cancers have reported such gene signatures. However, the overlap of gene signatures in the same disease has been limited so far, and some reported signatures have not been reproduced in other populations. Clearly, the methods used for verifying novel gene signatures need improvement. In this article, we describe an experiment in which microarrays and sample hybridization are designed according to the statistical principles of randomization, replication and blocking. Our results show that such designs provide unbiased estimation of differential expression levels as well as powerful tests for them.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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