An elegant miRror: microRNAs in stem cells,developmental timing and cancer

MicroRNAs (miRNAs) were first discovered in genetic screens for regulators of developmental timing in the stem-cell-like seam cell lineage in Caenorhabditis elegans. As members of the heterochronic pathway, the lin-4 and let-7 miRNAs are required in the seam cells for the correct progression of stage-specific events and to ensure that cell cycle exit and terminal differentiation occur at the correct time. Other heterochronic genes such as lin-28 and lin-41 are direct targets of the lin-4 and let-7 miRNAs. Recent findings on the functions of the let-7 and lin-4/mir-125 miRNA families and lin-28 and lin-41 orthologs from a variety of organisms suggest that core elements of the heterochronic pathway are retained in mammalian stem cells and development. In particular, these genes appear to form bistable switches via double-negative feedback loops in both nematode and mammalian stem cell development, the functional relevance of which is finally becoming clear. let-7 inhibits stem cell self-renewal in both normal and cancer stem cells of the breast and acts as a tumor suppressor in lung and breast cancer. let-7 also promotes terminal differentiation at the larval to adult transition in both nematode stem cells and fly wing imaginal discs and inhibits proliferation of human lung and liver cancer cells. Conversely, LIN-28 is a highly specific embryonic stem cell marker and is one of four “stemness” factors used to reprogram adult fibroblasts into induced pluripotent stem cells; furthermore, lin-28 is oncogenic in hepatocellular carcinomas. Therefore, a core module of heterochronic genes—lin-28, lin-41, let-7, and lin-4/mir-125—acts as an ancient regulatory switch for differentiation in stem cells (and in some cancers), illustrating that nematode seam cells mirror miRNA regulatory networks in mammalian stem cells during both normal development and cancer.     

Characterization and expression patterns of <Emphasis Type="Italic">let-7</Emphasis> microRNA in the silkworm (<Emphasis Type="Italic">Bombyx mori</Emphasis>)

Background  

lin-4 and let-7, the two founding members of heterochronic microRNA genes, are firstly confirmed in Caenorhabditis elegans to control the proper timing of developmental programs in a heterochronic pathway. let-7 has been thought to trigger the onset of adulthood across animal phyla. Ecdysone and Broad-Complex are required for the temporal expression of let-7 in Drosophila melanogaster. For a better understanding of the conservation and functions of let-7, we seek to explore how it is expressed in the silkworm (Bombyx mori).     

acn-1, a C. elegans homologue of ACE,genetically interacts with the let-7 microRNA and other heterochronic genes

The heterochronic pathway in C. elegans controls the relative timing of cell fate decisions during post-embryonic development. It includes a network of microRNAs (miRNAs), such as let-7, and protein-coding genes, such as the stemness factors, LIN-28 and LIN-41. Here we identified the acn-1 gene, a homologue of mammalian angiotensin-converting enzyme (ACE), as a new suppressor of the stem cell developmental defects of let-7 mutants. Since acn-1 null mutants die during early larval development, we used RNAi to characterize the role of acn-1 in C. elegans seam cell development, and determined its interaction with heterochronic factors, including let-7 and its downstream interactors – lin-41, hbl-1, and apl-1. We demonstrate that although RNAi knockdown of acn-1 is insufficient to cause heterochronic defects on its own, loss of acn-1 suppresses the retarded phenotypes of let-7 mutants and enhances the precocious phenotypes of hbl-1, though not lin-41, mutants. Conversely, the pattern of acn-1 expression, which oscillates during larval development, is disrupted by lin-41 mutants but not by hbl-1 mutants. Finally, we show that acn-1(RNAi) enhances the let-7-suppressing phenotypes caused by loss of apl-1, a homologue of the Alzheimer's disease-causing amyloid precursor protein (APP), while significantly disrupting the expression of apl-1 during the L4 larval stage. In conclusion, acn-1 interacts with heterochronic genes and appears to function downstream of let-7 and its target genes, including lin-41 and apl-1.     

Transcriptional changes in response to X chromosome dosage in the mouse: implications for X inactivation and the molecular basis of Turner Syndrome

Background

In many eukaryotes, microRNAs (miRNAs) bind to complementary sites in the 3'-untranslated regions (3'-UTRs) of target messenger RNAs (mRNAs) and regulate their expression at the stage of translation. Recent studies have revealed that many miRNAs are evolutionarily conserved; however, the evolution of their target genes has yet to be systematically characterized. We sought to elucidate a set of conserved miRNA/target-gene pairs and to analyse the mechanism underlying miRNA-mediated gene regulation in the early stage of bilaterian evolution.

Results

Initially, we extracted five evolutionarily conserved miRNAs (let-7, miR-1, miR-124, miR-125/lin-4, and miR-34) among five diverse bilaterian animals. Subsequently, we designed a procedure to predict evolutionarily conserved miRNA/target-gene pairs by introducing orthologous gene information. As a result, we extracted 31 orthologous miRNA/target-gene pairs that were conserved among at least four diverse bilaterian animals; the prediction set showed prominent enrichment of orthologous miRNA/target-gene pairs that were verified experimentally. Approximately 84% of the target genes were regulated by three miRNAs (let-7, miR-1, and miR-124) and their function was classified mainly into the following categories: development, muscle formation, cell adhesion, and gene regulation. We used a reporter gene assay to experimentally verify the downregulation of six candidate pairs (out of six tested pairs) in HeLa cells.

Conclusions

The application of our new method enables the identification of 31 miRNA/target-gene pairs that were expected to have been regulated from the era of the common bilaterian ancestor. The downregulation of all six candidate pairs suggests that orthologous information contributed to the elucidation of the primordial set of genes that has been regulated by miRNAs; it was also an efficient tool for the elimination of false positives from the predicted candidates. In conclusion, our study identified potentially important miRNA-target pairs that were evolutionarily conserved throughout diverse bilaterian animals and that may provide new insights into early-stage miRNA functions.     

High-Throughput Profiling of Caenorhabditis elegans Starvation-Responsive microRNAs

MicroRNAs (miRNAs) are non-coding RNAs of ~22 nucleotides in length that regulate gene expression by interfering with the stability and translation of mRNAs. Their expression is regulated during development, under a wide variety of stress conditions and in several pathological processes. In nature, animals often face feast or famine conditions. We observed that subjecting early L4 larvae from Caenorhabditis elegans to a 12-hr starvation period produced worms that are thinner and shorter than well-fed animals, with a decreased lipid accumulation, diminished progeny, reduced gonad size, and an increased lifespan. Our objective was to identify which of the 302 known miRNAs of C. elegans changed their expression under starvation conditions as compared to well-fed worms by means of deep sequencing in early L4 larvae. Our results indicate that 13 miRNAs (miR-34-3p, the family of miR-35-3p to miR-41-3p, miR-39-5p, miR-41-5p, miR-240-5p, miR-246-3p and miR-4813-5p) were upregulated, while 2 miRNAs (let-7-3p and miR-85-5p) were downregulated in 12-hr starved vs. well-fed early L4 larvae. Some of the predicted targets of the miRNAs that changed their expression in starvation conditions are involved in metabolic or developmental process. In particular, miRNAs of the miR-35 family were upregulated 6–20 fold upon starvation. Additionally, we showed that the expression of gld-1, important in oogenesis, a validated target of miR-35-3p, was downregulated when the expression of miR-35-3p was upregulated. The expression of another reported target, the cell cycle regulator lin-23, was unchanged during starvation. This study represents a starting point for a more comprehensive understanding of the role of miRNAs during starvation in C. elegans.     

Plasma microRNAs are associated with acute exacerbation in idiopathic pulmonary fibrosis

Background

Acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) has high short-term mortality with unknown causes. To predict this malignant condition in clinics is challenging. In this study, we aim to demonstrate whether there are miRNAs that differ between AE-IPF and stable IPF, which may be served as reliable biomarker for AE-IPF prediction.

Methods

Human fibrotic-associated miRNAs arrays were designed to detect miRNAs expression in plasma of 3 AE-IPF patients, 3 Stable-IPF (S-IPF) patients and 3 normal controls (NC). Differentially expressed miRNAs between AE-IPF and S-IPF patients were selected for further analyses. The validation studies were carried out in plasma of 12 AE-IPF patients, 45 S-IPF patients and 51 healthy control subjects. Signaling pathways and cellular processes interacted with validated miRNAs were predicted by DIANA-miRPath.

Results

According to the array analysis, 6 miRNAs showed differentiated expression between AE-IPF and S-IPF patients (P?<?0.05). In the validation studies, let-7d-5p was decreased in S-IPF and further decreased in AE-IPF, when compared to NC (0.0003?±?0.0002 vs 0.003?±?0.002, P?<?0.01 and 0.0007?±?0.0005 vs 0.003?±?0.002, P?<?0.01). While miR-25-3p was obviously decreased in S-IPF (0.0002?±?0.0001 vs 0.0003?±?0.0003, P?<?0.01) but significantly increased in AE-IP (0.0023?±?0.002 vs 0.0003?±?0.0003, P?<?0.01). In receiver-operator characteristic (ROC) curve analysis, the areas under the curve (AUCs) of miR-25-3p and let-7d-5p were 0.83 and 0.75, respectively. The sensitivity at fixed specificity of 90% was improved from 50% to 66.7% when the two miRNAs were combined. The functional prediction of miRNAs suggested that the loss of anti-fibrotic capacity and the gain of uncontrolled cell growth may be required in AE-IPF pathogenesis.

Conclusions

In conclusion, miR-25-3p and let-7d-5p in plasma were differentially expressed between AE-IPF and S-IPF. A combination of these two miRNAs may be a potential biomarker for AE-IPF from IPF.

     

Identification of microRNAs as potential prognostic markers in ependymoma

Introduction

We have examined expression of microRNAs (miRNAs) in ependymomas to identify molecular markers of value for clinical management. miRNAs are non-coding RNAs that can block mRNA translation and affect mRNA stability. Changes in the expression of miRNAs have been correlated with many human cancers.

Materials and Methods

We have utilized TaqMan Low Density Arrays to evaluate the expression of 365 miRNAs in ependymomas and normal brain tissue. We first demonstrated the similarity of expression profiles of paired frozen tissue (FT) and paraffin-embedded specimens (FFPE). We compared the miRNA expression profiles of 34 FFPE ependymoma samples with 8 microdissected normal brain tissue specimens enriched for ependymal cells. miRNA expression profiles were then correlated with tumor location, histology and other clinicopathological features.

Results

We have identified miRNAs that are over-expressed in ependymomas, such as miR-135a and miR-17-5p, and down-regulated, such as miR-383 and miR-485-5p. We have also uncovered associations between expression of specific miRNAs which portend a worse prognosis. For example, we have identified a cluster of miRNAs on human chromosome 14q32 that is associated with time to relapse. We also found that miR-203 is an independent marker for relapse compared to the parameters that are currently used. Additionally, we have identified three miRNAs (let-7d, miR-596 and miR-367) that strongly correlate to overall survival.

Conclusion

We have identified miRNAs that are differentially expressed in ependymomas compared with normal ependymal tissue. We have also uncovered significant associations of miRNAs with clinical behavior. This is the first report of clinically relevant miRNAs in ependymomas.     

The Caenorhabditis elegans hunchback-like gene lin-57/hbl-1 controls developmental time and is regulated by microRNAs

Temporal control of development is an important aspect of pattern formation that awaits complete molecular analysis. We identified lin-57 as a member of the C. elegans heterochronic gene pathway, which ensures that postembryonic developmental events are appropriately timed. Loss of lin-57 function causes the hypodermis to terminally differentiate and acquire adult character prematurely. lin-57 is hbl-1, revealing a role for the worm hunchback homolog in control of developmental time. Significantly, fly hunchback (hb) temporally specifies cell fates in the nervous system. The hbl-1/lin-57 3'UTR is required for postembryonic downregulation in the hypodermis and nervous system and contains multiple putative binding sites for temporally regulated microRNAs, including let-7. Indeed, we find that hbl-1/lin-57 is regulated by let-7, at least in the nervous system. Examination of the hb 3'UTR reveals potential binding sites for known fly miRNAs. Thus, evolutionary conservation of hunchback genes may include temporal control of cell fate specification and microRNA-mediated regulation.     

NMR structure of the let-7 miRNA interacting with the site LCS1 of lin-41 mRNA from Caenorhabditis elegans

We have determined the 3D structure of a 34-nt RNA construct, herein named LCS1co, which mimics the interaction of let-7 microRNA (miRNA) to one of its complementary binding sites, LCS1, in the 3′-untranslated region of lin-41 mRNA by solution-state NMR spectroscopy. let-7 miRNAs control the timing of development of the nematode Caenorhabditis elegans and are highly conserved in mammals. The sequence and structure of the two conserved let-7 complementary sites, LCS1 and LCS2, in the 3′-untranslated region of lin-41 mRNA are important for a proper downregulation of lin-41. The high-resolution NMR structure reveals details of the binding of let-7 miRNA to lin-41 mRNA which involves formation of a complex with non-canonical structural elements within the seed region. LCS1co exhibits a stem-loop structure with two stems, an asymmetric internal loop and an adenine bulge. Comparison with the NMR solution-state structure of the let-7:lin-41 complex involving the LCS2-binding site shows that conformational freedom of the asymmetric internal loop of LCS1co correlates with a smaller bend between the upper and lower stems in comparison to the well-defined asymmetric loop of LCS2co.     

Functional relevance of “seed” and “non-seed” sequences in microRNA-mediated promotion of C. elegans developmental progression

The founding heterochronic microRNAs, lin-4 and let-7, together with their validated targets and well-characterized phenotypes in C. elegans, offer an opportunity to test functionality of microRNAs in a developmental context. In this study, we defined sequence requirements at the microRNA level for these two microRNAs, evaluating lin-4 and let-7 mutant microRNAs for their ability to support temporal development under conditions where the wild-type lin-4 and let-7 gene products are absent. For lin-4, we found a strong requirement for seed sequences, with function drastically affected by several central mutations in the seed sequence, while rescue was retained by a set of mutations peripheral to the seed. let-7 rescuing activity was retained to a surprising degree by a variety of central seed mutations, while several non-seed mutant effects support potential noncanonical contributions to let-7 function. Taken together, this work illustrates both the functional partnership between seed and non-seed sequences in mediating C. elegans temporal development and a diversity among microRNA effectors in the contributions of seed and non-seed regions to activity.     

Identification of specific let-7 microRNA binding complexes in Caenorhabditis elegans

Little is known about the protein complexes required for microRNA formation and function. Here we used native gel electrophoresis to identify miRNA ribonucleoprotein complexes (miRNPs) in Caenorhabditis elegans. Our data reveal multiple distinct miRNPs that assemble on the let-7 miRNA in vitro. The formation of these complexes is affected but not abolished by alg-1 or alg-2 null mutations. The largest complex (M*) with an estimated molecular mass of >669 kDa cofractionates with the known RISC factors ALG-1, VIG-1, and TSN-1. The M* complex and two complexes, M3 and M4, with similar molecular weights of ~500 kDa, also assemble on all other miRNAs used in our experiments. Two smaller complexes, M1 (~160 kDa) and M2 (~250 kDa), assemble on the members of the let-7 miRNAs family but not lin-4 or mir-234, and their formation is highly dependent on specific sequences in the 5′ seed region of let-7. Moreover, an unidentified protein, p40, which only appears in the M1 and M2 complexes, was detected by UV triggered cross-linking to let-7 but not to lin-4. The cross-linking of p40 to let-7 is also dependent on the let-7 sequence. Another unidentified protein, p13, is detected in all let-7 binding complexes and lin-4 cross-linked products. Our data suggest that besides being present in certain large miRNPs with sizes similar to reported RISC, the let-7 miRNA also assembles with specific binding proteins and forms distinct small complexes.     

Uncovering new functions for microRNAs in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, microRNA (miRNA) regulation of development was first observed in the striking abnormalities of lin-4 and let-7 loss of function mutants. However, after these first two miRNA mutant phenotypes were described, progress on the identification of miRNA functions in worms slowed considerably. Recent advances reveal new functions for miRNAs in embryonic and larval development as well as in the regulation of lifespan and stress response. Results from a combination of?computational, biochemical, and genetic approaches have deepened our understanding of miRNA regulation of target mRNAs and support the hypothesis that miRNAs have an important role in ensuring the robustness of developmental and physiological pathways.     

Circulating Exosomal microRNAs as Biomarkers of Colon Cancer

Purpose

Exosomal microRNAs (miRNAs) have been attracting major interest as potential diagnostic biomarkers of cancer. The aim of this study was to characterize the miRNA profiles of serum exosomes and to identify those that are altered in colorectal cancer (CRC). To evaluate their use as diagnostic biomarkers, the relationship between specific exosomal miRNA levels and pathological changes of patients, including disease stage and tumor resection, was examined.

Experimental Design

Microarray analyses of miRNAs in exosome-enriched fractions of serum samples from 88 primary CRC patients and 11 healthy controls were performed. The expression levels of miRNAs in the culture medium of five colon cancer cell lines were also compared with those in the culture medium of a normal colon-derived cell line. The expression profiles of miRNAs that were differentially expressed between CRC and control sample sets were verified using 29 paired samples from post-tumor resection patients. The sensitivities of selected miRNAs as biomarkers of CRC were evaluated and compared with those of known tumor markers (CA19-9 and CEA) using a receiver operating characteristic analysis. The expression levels of selected miRNAs were also validated by quantitative real-time RT-PCR analyses of an independent set of 13 CRC patients.

Results

The serum exosomal levels of seven miRNAs (let-7a, miR-1229, miR-1246, miR-150, miR-21, miR-223, and miR-23a) were significantly higher in primary CRC patients, even those with early stage disease, than in healthy controls, and were significantly down-regulated after surgical resection of tumors. These miRNAs were also secreted at significantly higher levels by colon cancer cell lines than by a normal colon-derived cell line. The high sensitivities of the seven selected exosomal miRNAs were confirmed by a receiver operating characteristic analysis.

Conclusion

Exosomal miRNA signatures appear to mirror pathological changes of CRC patients and several miRNAs are promising biomarkers for non-invasive diagnosis of the disease.