Regulatory networks and 5' partner usage of miRNA host gene fusions in breast cancer.

Genomic rearrangements in cancer cells can create gene fusions where the juxtaposition of two different genes leads to the production of chimeric proteins or altered gene expression through promoter-swapping. We have previously shown that fusion transcripts involving microRNA (miRNA) host genes contribute to deregulation of miRNA expression regardless of the protein-coding potential of these transcripts. Many different genes can also be used as 5' partners by a miRNA host gene in what we named recurrent miRNA-convergent fusions. Here, we have explored the properties of 5' partners in fusion transcripts that involve miRNA hosts in breast tumours from The Cancer Genome Atlas (TCGA). We hypothesised that firstly, 5' partner genes should belong to pathways and transcriptional programmes that reflect the tumour phenotype and secondly, there should be a selection for fusion events that shape miRNA expression to benefit the tumour cell through the known hallmarks of cancer. We found that the set of 5' partners in miRNA host fusions is non-random, with overrepresentation of highly expressed genes in pathways active in cancer including epithelial-to-mesenchymal transition, translational regulation and oestrogen signalling. Furthermore, many miRNAs were upregulated in samples with host gene fusions, including established oncogenic miRNAs such as mir-21 and the mir-106b~mir-93~mir-25 cluster. To the list of mechanisms for deregulation of miRNA expression, we have added fusion transcripts that change the promoter region. We propose that this adds material for genetic selection and tumour evolution in cancer cells and that miRNA host fusions can act as tumour 'drivers'.

Analysis of fusion transcripts indicates widespread deregulation of snoRNAs and their host genes in breast cancer.

Genomic rearrangements in cancer can join the sequences of two separate genes. Studies of such gene fusion events have mainly focused on identification of fusion proteins from the chimeric transcripts. We have previously investigated how fusions instead can affect the expression of intronic microRNA (miRNA) genes that are encoded within fusion gene partners. Here, we extend our analysis to small nucleolar RNAs (snoRNAs) that also are embedded within protein-coding or noncoding host genes. We found that snoRNA hosts are selectively enriched in fusion transcripts, like miRNA host genes, and that this enrichment is associated with all snoRNA classes. These structural changes may have functional consequences for the cell; proteins involved in the protein translation machinery are overrepresented among snoRNA host genes, a gene architecture assumed to be needed for closely coordinated expression of snoRNAs and host proteins. Our data indicate that this structure is frequently disrupted in cancer. We furthermore observed that snoRNA genes involved in fusions tend to associate with stronger promoters than the natural host, suggesting a mechanism that selects for snoRNA overexpression. In summary, we highlight a previously unexplored frequent structural change in cancer that affects important components of cellular physiology.

Refinement of breast cancer molecular classification by miRNA expression profiles.

BACKGROUND: Accurate classification of breast cancer using gene expression profiles has contributed to a better understanding of the biological mechanisms behind the disease and has paved the way for better prognostication and treatment prediction. RESULTS: We found that miRNA profiles largely recapitulate intrinsic subtypes. In the case of HER2-enriched tumors a small set of miRNAs including the HER2-encoded mir-4728 identifies the group with very high specificity. We also identified differential expression of the miR-99a/let-7c/miR-125b miRNA cluster as a marker for separation of the Luminal A and B subtypes. High expression of this miRNA cluster is linked to better overall survival among patients with Luminal A tumors. Correlation between the miRNA cluster and their precursor LINC00478 is highly significant suggesting that its expression could help improve the accuracy of present day's signatures. CONCLUSIONS: We show here that miRNA expression can be translated into mRNA profiles and that the inclusion of miRNA information facilitates the molecular diagnosis of specific subtypes, in particular the clinically relevant sub-classification of luminal tumors.

PAPD5-mediated 3' adenylation and subsequent degradation of miR-21 is disrupted in proliferative disease.

Next-generation sequencing experiments have shown that microRNAs (miRNAs) are expressed in many different isoforms (isomiRs), whose biological relevance is often unclear. We found that mature miR-21, the most widely researched miRNA because of its importance in human disease, is produced in two prevalent isomiR forms that differ by 1 nt at their 3' end, and moreover that the 3' end of miR-21 is posttranscriptionally adenylated by the noncanonical poly(A) polymerase PAPD5. PAPD5 knockdown caused an increase in the miR-21 expression level, suggesting that PAPD5-mediated adenylation of miR-21 leads to its degradation. Exoribonuclease knockdown experiments followed by small-RNA sequencing suggested that PARN degrades miR-21 in the 3'-to-5' direction. In accordance with this model, microarray expression profiling demonstrated that PAPD5 knockdown results in a down-regulation of miR-21 target mRNAs. We found that disruption of the miR-21 adenylation and degradation pathway is a general feature in tumors across a wide range of tissues, as evidenced by data from The Cancer Genome Atlas, as well as in the noncancerous proliferative disease psoriasis. We conclude that PAPD5 and PARN mediate degradation of oncogenic miRNA miR-21 through a tailing and trimming process, and that this pathway is disrupted in cancer and other proliferative diseases.

Mining of public sequencing databases supports a non-dietary origin for putative foreign miRNAs: underestimated effects of contamination in NGS.

The report that exogenous plant miRNAs are able to cross the mammalian gastrointestinal tract and exert gene-regulation mechanism in mammalian tissues has yielded a lot of controversy, both in the public press and the scientific literature. Despite the initial enthusiasm, reproducibility of these results was recently questioned by several authors. To analyze the causes of this unease, we searched for diet-derived miRNAs in deep-sequencing libraries performed by ourselves and others. We found variable amounts of plant miRNAs in publicly available small RNA-seq data sets of human tissues. In human spermatozoa, exogenous RNAs reached extreme, biologically meaningless levels. On the contrary, plant miRNAs were not detected in our sequencing of human sperm cells, which was performed in the absence of any known sources of plant contamination. We designed an experiment to show that cross-contamination during library preparation is a source of exogenous RNAs. These contamination-derived exogenous sequences even resisted oxidation with sodium periodate. To test the assumption that diet-derived miRNAs were actually contamination-derived, we sought in the literature for previous sequencing reports performed by the same group which reported the initial finding. We analyzed the spectra of plant miRNAs in a small RNA sequencing study performed in amphioxus by this group in 2009 and we found a very strong correlation with the plant miRNAs which they later reported in human sera. Even though contamination with exogenous sequences may be easy to detect, cross-contamination between samples from the same organism can go completely unnoticed, possibly affecting conclusions derived from NGS transcriptomics.

Passenger strand loading in overexpression experiments using microRNA mimics.

MicroRNAs (miRNAs) are important regulators of gene function and manipulation of miRNAs is a central component of basic research. Modulation of gene expression by miRNA gain-of-function can be based on different approaches including transfection with miRNA mimics; artificial, chemically modified miRNA-like small RNAs. These molecules are intended to mimic the function of a miRNA guide strand while bypassing the maturation steps of endogenous miRNAs. Due to easy accessibility through commercial providers this approach has gained popularity, and accuracy is often assumed without prior independent testing. Our in silico analysis of over-represented sequence motifs in microarray expression data and sequencing of AGO-associated small RNAs indicate, however, that miRNA mimics may be associated with considerable side-effects due to the unwanted activity of the miRNA mimic complementary strand.

Comparative microRNA profiling of Trypanosoma cruzi infected human cells.

Introduction: Trypanosoma cruzi, the causative agent of Chagas disease, can infect almost any nucleated cell in the mammalian host. Although previous studies have described the transcriptomic changes that occur in host cells during parasite infection, the understanding of the role of post-transcriptional regulation in this process is limited. MicroRNAs, a class of short non-coding RNAs, are key players in regulating gene expression at the post-transcriptional level, and their involvement in the host-T. cruzi interplay is a growing area of research. However, to our knowledge, there are no comparative studies on the microRNA changes that occur in different cell types in response to T. cruzi infection. Methods and results: Here we investigated microRNA changes in epithelial cells, cardiomyocytes and macrophages infected with T. cruzi for 24 hours, using small RNA sequencing followed by careful bioinformatics analysis. We show that, although microRNAs are highly cell type-specific, a signature of three microRNAs -miR-146a, miR-708 and miR-1246, emerges as consistently responsive to T. cruzi infection across representative human cell types. T. cruzi lacks canonical microRNA-induced silencing mechanisms and we confirm that it does not produce any small RNA that mimics known host microRNAs. We found that macrophages show a broad response to parasite infection, while microRNA changes in epithelial and cardiomyocytes are modest. Complementary data indicated that cardiomyocyte response may be greater at early time points of infection. Conclusions: Our findings emphasize the significance of considering microRNA changes at the cellular level and complement previous studies conducted at higher organizational levels, such as heart samples. While miR-146a has been previously implicated in T. cruzi infection, similarly to its involvement in many other immunological responses, miR-1246 and miR-708 are demonstrated here for the first time. Given their expression in multiple cell types, we anticipate our work as a starting point for future investigations into their role in the post-transcriptional regulation of T. cruzi infected cells and their potential as biomarkers for Chagas disease.

Search for an aetiological virus candidate in chronic lymphocytic leukaemia by extensive transcriptome analysis.

As an approach to determining the aetiology of chronic lymphocytic leukaemia (CLL), we searched for a virus expressed in human CLL B-cells by combining high-throughput sequencing and digital subtraction. Pooled B-cell mRNA transcriptomes from five CLL patients and five healthy donors were sequenced with 454 Life Sciences technology. Human reads were excluded by BLAST (Basic Local Alignment Search Tool) and BLAT (BLAST-like alignment tool) searches. Remaining reads were screened with BLAST against viral databases. Purified B-cells from two CLL patients, with and without stimulation by phorbol-esters, were sequenced using Illumina technology to achieve depth of sequencing. Burrows-Wheeler Aligner mapping and BLAST searches were used for the Illumina data. Pyrosequencing resulted in about 400 000 reads per sample. No viral candidate could be found. Illumina single-end sequencing for 115 cycles yielded an average of 26 ± 2·5 million filtered reads per sample, of which 2·2 ± 0·6 million remained unmapped to human references. BLAST searches of these reads against viral and human databases assigned nine reads to an Epstein-Barr virus origin, in one sample following phorbol-ester stimulation. Other reads showing a putative viral origin were dismissed after further analysis. Despite an in-depth analysis of the CLL transcriptome reaching more than 100 million sequences, we have not found evidence for a putative viral candidate in CLL.

Expression of MicroRNAs Is Dysregulated by HIV While Mycobacterium tuberculosis Drives Alterations of Small Nucleolar RNAs in HIV Positive Adults With Active Tuberculosis.

HIV infection affects the course of tuberculosis (TB), and HIV and Mycobacterium tuberculosis (Mtb) synergize in disease progression through complex immunological interplay. To gain further understanding of these mechanisms, we compared the microRNA (miRNA) and small nucleolar RNA (snoRNA) expression patterns in whole blood of individuals with active TB, with and without HIV coinfection (HIV+/TB+ and HIV-/TB+), and HIV and TB-negative individuals (HIV-/TB-). We found that 218 miRNAs were differentially expressed between HIV+/TB+ and HIV-/TB+, while no statistically significant difference in snoRNA expression was observed between these groups. In contrast, both miRNA (n = 179) and snoRNA (n = 103) expression patterns were significantly altered in HIV+/TB+ individuals compared to those of the HIV-/TB- controls. Of note, 26 of these snoRNAs were also significantly altered between the HIV-/TB+ and HIV-/TB- groups. Normalization toward the miRNA and snoRNA expression patterns of the HIV-/TB- control group was noted during anti-TB and antiretroviral treatment in HIV+/TB+ participants. In summary, these results show that HIV coinfection influences miRNA expression in active TB. In contrast, snoRNA expression patterns differ between individuals with and without active TB, independently of HIV coinfection status. Moreover, in coinfected individuals, therapy-induced control of HIV replication and clearance of Mtb appears to normalize the expression of some small non-coding RNA (sncRNA). These findings suggest that dysregulation of miRNA is a mechanism by which HIV may modify immunity against TB, while active TB alters snoRNA expression. Improved understanding of how regulation of sncRNA expression influences the disease course in coinfected individuals may have implications for diagnostics, risk stratification, and host-directed therapy. Here, we propose a novel mechanism by which HIV alters the immune response to TB.

MicroRNAs and other small silencing RNAs in cancer.

Small noncoding RNAs are key controllers of cellular function, and their deregulation can lead to cancer development and metastatic evolution. This review summarizes the most important examples of small RNAs involved in human cancer and discusses their clinical use as biomarkers and drug targets for diagnosis, prognosis, and treatment of cancer. We also describe the possible mechanisms underlying small RNA-mediated transformation and outline the future describing new small RNA families with great potential in cancer biology.

Fine-tuning the metabolic rewiring and adaptation of translational machinery during an epithelial-mesenchymal transition in breast cancer cells.

BACKGROUND: During breast cancer progression, the epithelial to mesenchymal transition has been associated with metastasis and endocrine therapy resistance; however, the underlying mechanisms remain elusive. To gain insight into this process, we studied the transition undergone by MCF7-derived cells, which is driven by the constitutive nuclear expression of a MKL1 variant devoid of the actin-binding domain (MKL1 ΔN200). We characterized the adaptive changes that occur during the MKL1-induced cellular model and focused on regulation of translation machinery and metabolic adaptation. METHODS: We performed a genome-wide analysis at the transcriptional and translational level using ribosome profiling complemented with RNA-Seq and analyzed the expression of components of the translation machinery and enzymes involved in energy metabolism. NGS data were correlated with metabolomic measurements and quantification of specific mRNAs extracted from polysomes and western blots. RESULTS: Our results reveal the expression profiles of a luminal to basal-like state in accordance with an epithelial to mesenchymal transition. During the transition, the synthesis of ribosomal proteins and that of many translational factors was upregulated. This overexpression of the translational machinery appears to be regulated at the translational level. Our results indicate an increase of ribosome biogenesis and translation activity. We detected an extensive metabolic rewiring occurring in an already "Warburg-like" context, in which enzyme isoform switches and metabolic shunts indicate a crucial role of HIF-1α along with other master regulatory factors. Furthermore, we detected a decrease in the expression of enzymes involved in ribonucleotide synthesis from the pentose phosphate pathway. During this transition, cells increase in size, downregulate genes associated with proliferation, and strongly upregulate expression of cytoskeletal and extracellular matrix genes. CONCLUSIONS: Our study reveals multiple regulatory events associated with metabolic and translational machinery adaptation during an epithelial mesenchymal-like transition process. During this major cellular transition, cells achieve a new homeostatic state ensuring their survival. This work shows that ribosome profiling complemented with RNA-Seq is a powerful approach to unveil in-depth global adaptive cellular responses and the interconnection among regulatory circuits, which will be helpful for identification of new therapeutic targets.

MiRNA expression in urothelial carcinomas: important roles of miR-10a, miR-222, miR-125b, miR-7 and miR-452 for tumor stage and metastasis, and frequent homozygous losses of miR-31.

We analyzed 34 cases of urothelial carcinomas by miRNA, mRNA and genomic profiling. Unsupervised hierarchical clustering using expression information for 300 miRNAs produced 3 major clusters of tumors corresponding to Ta, T1 and T2-T3 tumors, respectively. A subsequent SAM analysis identified 51 miRNAs that discriminated the 3 pathological subtypes. A score based on the expression levels of the 51 miRNAs, identified muscle invasive tumors with high precision and sensitivity. MiRNAs showing high expression in muscle invasive tumors included miR-222 and miR-125b and in Ta tumors miR-10a. A miRNA signature for FGFR3 mutated cases was also identified with miR-7 as an important member. MiR-31, located in 9p21, was found to be homozygously deleted in 3 cases and miR-452 and miR-452* were shown to be over expressed in node positive tumors. In addition, these latter miRNAs were shown to be excellent prognostic markers for death by disease as outcome. The presented data shows that pathological subtypes of urothelial carcinoma show distinct miRNA gene expression signatures.

HER2 and p95HER2 differentially regulate miRNA expression in MCF-7 breast cancer cells and downregulate MYB proteins through miR-221/222 and miR-503.

The HER2 oncogene and its truncated form p95HER2 play central roles in breast cancer. Here, we show that although HER2 and p95HER2 generally elicit qualitatively similar changes in miRNA profile in MCF-7 breast cancer cells, a subset of changes are distinct and p95HER2 shifts the miRNA profile towards the basal breast cancer subtype. High-throughput miRNA profiling was carried out 15, 36 and 60 h after HER2 or p95HER2 expression and central hits validated by RT-qPCR. miRNAs strongly regulated by p95HER2 yet not by HER2, included miR-221, miR-222, miR-503, miR-29a, miR-149, miR-196 and miR-361. Estrogen receptor-α (ESR1) expression was essentially ablated by p95HER2 expression, in a manner recapitulated by miR-221/-222 mimics. c-Myb family transcription factors MYB and MYBL1, but not MYBL2, were downregulated by p95HER2 and by miR-503 or miR-221/-222 mimics. MYBL1 3'UTR inhibition by miR-221/222 was lost by deletion of a single putative miR-221/222 binding sites. p95HER2 expression, or knockdown of either MYB protein, elicited upregulation of tissue inhibitor of matrix metalloprotease-2 (TIMP2). miR-221/222 and -503 mimics increased, and TIMP2 knockdown decreased, cell migration and invasion. A similar pathway was operational in T47D- and SKBr-3 cells. This work reveals important differences between HER2- and p95HER2- mediated miRNA changes in breast cancer cells, provides novel mechanistic insight into regulation of MYB family transcription factors by p95HER2, and points to a role for a miR-221/222- MYB family-TIMP2 axis in regulation of motility in breast cancer cells.

miRNA-convergent gene fusions.

In a recent study published in Nature Communications, we showed that intron-encoded microRNA genes (miRNA) are frequent partners of fusion genes in the cancer genome. Analyzed from a functional rather than structural perspective, these rearrangements represent a new class of fusions we called "miRNA-convergent fusions".

Non-coding RNA fragments account for the majority of annotated piRNAs expressed in somatic non-gonadal tissues.

PIWI-interacting RNAs (piRNAs) are regarded as the guardians of the genome because they tackle genome stability-threatening transposable elements in the germline. Recently, piRNAs were also reported in other types of cells, including mouse brain, malignant and non-malignant somatic tissues, and human plasma. This suggests that piRNA function might be broader than previously expected. Here, we show that different piRNA databases contain a subset of sequences that correspond to piRNA-sized fragments of ncRNAs (rRNAs, tRNAs, YRNAs, snRNAs, and snoRNAs) and intermediates of miRNA biogenesis. We discuss that the biogenesis of these sequences is probably independent of the PIWI pathway, and can therefore be considered contaminants in piRNA databases. Although a minority of annotated piRNAs falls in this category, they account for the vast majority of piRNA expression in somatic non-gonadal tissues. Since ncRNA fragments are ubiquitous and abundant, their confusion with piRNAs strongly impacts the estimation of piRNA expression outside of mammalian gonads.

A validation and extended description of the Lund taxonomy for urothelial carcinoma using the TCGA cohort.

Global gene expression analysis has been a major tool for urothelial carcinoma subtype discovery. This approach has revealed extensive complexity both in intrinsic features of the tumor cells and in the microenvironment. However, global gene expression cannot distinguish between gene expression signals originating from the tumor cells proper and from normal cells in the biopsy. Here, we use a large cohort of advanced urothelial carcinomas for which both gene expression data and extensive immunohistochemistry are available to create a supervised mRNA expression centroid classifier. This classifier identifies the major Lund taxonomy tumor cell phenotypes as defined by IHC. We apply this classifier to the independent TCGA dataset and show excellent associations between identified subtypes and genomic features. We validate a progressed version of Urothelial-like A (UroA-Prog) that shows FGFR3 mutations and CDKN2A deletions, and we show that the variant Urothelial-like C is almost devoid of FGFR3 mutations. We show that Genomically Unstable tumors are very distinct from Urothelial-like tumors at the genomic level, and that tumors classified as Basal/SCC-like all complied with the established definition for Basal/SCC-like tumors. We identify the Mesenchymal-like and Small-cell/Neuroendocrine-like subtypes, and demonstrate that patients with UroB and Sc/NE-like tumors show the worst overall survival.

Non-coding antisense transcription detected by conventional and single-stranded cDNA microarray.

BACKGROUND: Recent studies revealed that many mammalian protein-coding genes also transcribe their complementary strands. This phenomenon raises questions regarding the validity of data obtained from double-stranded cDNA microarrays since hybridization to both strands may occur. Here, we wanted to analyze experimentally the incidence of antisense transcription in human cells and to estimate their influence on protein coding expression patterns obtained by double-stranded microarrays. Therefore, we profiled transcription of sense and antisense independently by using strand-specific cDNA microarrays. RESULTS: Up to 88% of expressed protein coding loci displayed concurrent expression from the complementary strand. Antisense transcription is cell specific and showed a strong tendency to be positively correlated to the expression of the sense counterparts. Even if their expression is wide-spread, detected antisense signals seem to have a limited distorting effect on sense profiles obtained with double-stranded probes. CONCLUSION: Antisense transcription in humans can be far more common than previously estimated. However, it has limited influence on expression profiles obtained with conventional cDNA probes. This can be explained by a biological phenomena and a bias of the technique: a) a co-ordinate sense and antisense expression variation and b) a bias for sense-hybridization to occur with more efficiency, presumably due to variable exonic overlap between antisense transcripts.

Translational machinery and protein folding: evidence of conformational variants of the estrogen receptor alpha.

As an approach to understand how translation may affect protein folding, we analyzed structural and functional properties of the human estrogen receptor alpha synthesized by different eukaryotic translation systems. A minimum of three conformations of the receptor were detected using limited proteolysis and a sterol ligand-binding assay. The receptor in vitro translated in rabbit reticulocyte lysate was rapidly degraded by protease, produced major bands of about 34kDa and showed a high affinity for estradiol. In a wheat germ translation system, the receptor was more slowly digested. Two soluble co-existing conformations were evident by different degradation patterns and estradiol binding. Our data show that differences in the translation machinery may result in alternative conformations of the receptor with distinct sterol binding properties. These studies suggest that components of the cellular translation machinery itself might influence the protein folding pathways and the relative abundance of different receptor conformers.

Preparation of highly multiplexed small RNA sequencing libraries.

MicroRNAs (miRNAs) are ~22-nucleotide-long small non-coding RNAs that regulate the expression of protein-coding genes by base pairing to partially complementary target sites, preferentially located in the 3´ untranslated region (UTR) of target mRNAs. The expression and function of miRNAs have been extensively studied in human disease, as well as the possibility of using these molecules as biomarkers for prognostication and treatment guidance. To identify and validate miRNAs as biomarkers, their expression must be screened in large collections of patient samples. Here, we develop a scalable protocol for the rapid and economical preparation of a large number of small RNA sequencing libraries using dual indexing for multiplexing. Combined with the use of off-the-shelf reagents, more samples can be sequenced simultaneously on large-scale sequencing platforms at a considerably lower cost per sample. Sample preparation is simplified by pooling libraries prior to gel purification, which allows for the selection of a narrow size range while minimizing sample variation. A comparison with publicly available data from benchmarking of miRNA analysis platforms showed that this method captures absolute and differential expression as effectively as commercially available alternatives.