Open-ST: High-resolution spatial transcriptomics in 3D.

Spatial transcriptomics (ST) methods unlock molecular mechanisms underlying tissue development, homeostasis, or disease. However, there is a need for easy-to-use, high-resolution, cost-efficient, and 3D-scalable methods. Here, we report Open-ST, a sequencing-based, open-source experimental and computational resource to address these challenges and to study the molecular organization of tissues in 2D and 3D. In mouse brain, Open-ST captured transcripts at subcellular resolution and reconstructed cell types. In primary head-and-neck tumors and patient-matched healthy/metastatic lymph nodes, Open-ST captured the diversity of immune, stromal, and tumor populations in space, validated by imaging-based ST. Distinct cell states were organized around cell-cell communication hotspots in the tumor but not the metastasis. Strikingly, the 3D reconstruction and multimodal analysis of the metastatic lymph node revealed spatially contiguous structures not visible in 2D and potential biomarkers precisely at the 3D tumor/lymph node boundary. All protocols and software are available at https://rajewsky-lab.github.io/openst.

Epigenomic landscape of human colorectal cancer unveils an aberrant core of pan-cancer enhancers orchestrated by YAP/TAZ.

Cancer is characterized by pervasive epigenetic alterations with enhancer dysfunction orchestrating the aberrant cancer transcriptional programs and transcriptional dependencies. Here, we epigenetically characterize human colorectal cancer (CRC) using de novo chromatin state discovery on a library of different patient-derived organoids. By exploring this resource, we unveil a tumor-specific deregulated enhancerome that is cancer cell-intrinsic and independent of interpatient heterogeneity. We show that the transcriptional coactivators YAP/TAZ act as key regulators of the conserved CRC gained enhancers. The same YAP/TAZ-bound enhancers display active chromatin profiles across diverse human tumors, highlighting a pan-cancer epigenetic rewiring which at single-cell level distinguishes malignant from normal cell populations. YAP/TAZ inhibition in established tumor organoids causes extensive cell death unveiling their essential role in tumor maintenance. This work indicates a common layer of YAP/TAZ-fueled enhancer reprogramming that is key for the cancer cell state and can be exploited for the development of improved therapeutic avenues.

Characterization of Transcription Termination-Associated RNAs: New Insights into their Biogenesis, Tailing, and Expression in Primary Tumors.

Next-generation sequencing has uncovered novel classes of small RNAs (sRNAs) in eukaryotes, in addition to the well-known miRNAs, siRNAs, and piRNAs. In particular, sRNA species arise from transcription start sites (TSSs) and the transcription termination sites (TTSs) of genes. However, a detailed characterization of these new classes of sRNAs is still lacking. Here, we present a comprehensive study of sRNAs derived from TTSs of expressed genes (TTSa-RNAs) in human cell lines and primary tissues. Taking advantage of sRNA-sequencing, we show that TTSa-RNAs are present in the nuclei of human cells, are loaded onto both AGO1 and AGO2, and their biogenesis does not require DICER and AGO2 endonucleolytic activity. TTSa-RNAs display a strong bias against a G residue in the first position at 5' end, a known feature of AGO-bound sRNAs, and a peculiar oligoA tail at 3' end. AGO-bound TTSa-RNAs derive from genes involved in cell cycle progression regulation and DNA integrity checkpoints. Finally, we provide evidence that TTSa-RNAs can be detected by sRNA-Seq in primary human tissue, and their expression increases in tumor samples as compared to nontumor tissues, suggesting that in the future, TTSa-RNAs might be explored as biomarker for diagnosis or prognosis of human malignancies.

The oncogenic role of circPVT1 in head and neck squamous cell carcinoma is mediated through the mutant p53/YAP/TEAD transcription-competent complex.

BACKGROUND: Circular RNAs are a class of endogenous RNAs with various functions in eukaryotic cells. Worthy of note, circular RNAs play a critical role in cancer. Currently, nothing is known about their role in head and neck squamous cell carcinoma (HNSCC). The identification of circular RNAs in HNSCC might become useful for diagnostic and therapeutic strategies in HNSCC. RESULTS: Using samples from 115 HNSCC patients, we find that circPVT1 is over-expressed in tumors compared to matched non-tumoral tissues, with particular enrichment in patients with TP53 mutations. circPVT1 up- and down-regulation determine, respectively, an increase and a reduction of the malignant phenotype in HNSCC cell lines. We show that circPVT1 expression is transcriptionally enhanced by the mut-p53/YAP/TEAD complex. circPVT1 acts as an oncogene modulating the expression of miR-497-5p and genes involved in the control of cell proliferation. CONCLUSIONS: This study shows the oncogenic role of circPVT1 in HNSCC, extending current knowledge about the role of circular RNAs in cancer.

TP53 regulates miRNA association with AGO2 to remodel the miRNA-mRNA interaction network.

DNA damage activates TP53-regulated surveillance mechanisms that are crucial in suppressing tumorigenesis. TP53 orchestrates these responses directly by transcriptionally modulating genes, including microRNAs (miRNAs), and by regulating miRNA biogenesis through interacting with the DROSHA complex. However, whether the association between miRNAs and AGO2 is regulated following DNA damage is not yet known. Here, we show that, following DNA damage, TP53 interacts with AGO2 to induce or reduce AGO2's association of a subset of miRNAs, including multiple let-7 family members. Furthermore, we show that specific mutations in TP53 decrease rather than increase the association of let-7 family miRNAs, reducing their activity without preventing TP53 from interacting with AGO2. This is consistent with the oncogenic properties of these mutants. Using AGO2 RIP-seq and PAR-CLIP-seq, we show that the DNA damage-induced increase in binding of let-7 family members to the RISC complex is functional. We unambiguously determine the global miRNA-mRNA interaction networks involved in the DNA damage response, validating them through the identification of miRNA-target chimeras formed by endogenous ligation reactions. We find that the target complementary region of the let-7 seed tends to have highly fixed positions and more variable ones. Additionally, we observe that miRNAs, whose cellular abundance or differential association with AGO2 is regulated by TP53, are involved in an intricate network of regulatory feedback and feedforward circuits. TP53-mediated regulation of AGO2-miRNA interaction represents a new mechanism of miRNA regulation in carcinogenesis.

The role of TP53 in miRNA loading onto AGO2 and in remodelling the miRNA-mRNA interaction network.

BACKGROUND: DNA damage transactivates tumour protein p53 (TP53)-regulated surveillance, crucial in suppressing tumorigenesis. TP53 mediates this process directly by transcriptionally modulating gene and microRNA (miRNA) expression and indirectly by regulating miRNA biogenesis. However, the role of TP53 in regulating miRNA-AGO2 loading and global changes in AGO2 binding to its gene targets in response to DNA damage are unknown. These processes might be novel mechanisms by which TP53 regulates miRNAs in response to DNA damage. METHODS: To show the network of miRNA-mRNA interactions that occur in response to DNA damage, we stimulated TP53 wild-type and null cell-lines with doxorubicin and performed RNA sequencing from total RNA (RNA-Seq) and AGO2-immunoprecipitated RNA (AGO2-RIP-Seq). We used a combined AGO2 RIP-seq and AGO2 PAR-CLIP-seq (photoactivatable-ribonucleoside-enhanced cross-linking and immunoprecipitation) approach to determine the exact sites of interaction between the AGO2-bound miRNAs and their mRNA targets. FINDINGS: TP53 directly associated with AGO2, and induced and reduced loading of a subset of miRNAs, including the lethal 7 (let-7) miRNA family members, onto AGO2 in response to DNA damage. Although mutated TP53 maintained its capacity to interact with AGO2, it mediated unloading instead of loading of let-7 family miRNAs, thereby reducing their activity. We determined the miRNA-mRNA interaction networks involved in the response to DNA damage both in the presence and absence of TP53. Furthermore, we showed that miRNAs whose cellular abundance or differential loading onto AGO2 was regulated by TP53 were involved in an intricate network of regulatory feedback and feedforward circuits that fine-tuned gene expression levels in response to DNA damage to permit the repair of DNA damage or initiation of programmed cell death. INTERPRETATION: Control of AGO2 loading by TP53 is a new mechanism of miRNA regulation in carcinogenesis. FUNDING: UK Medical Research Council, Action Against Cancer.

PVT1: a rising star among oncogenic long noncoding RNAs.

It is becoming increasingly clear that short and long noncoding RNAs critically participate in the regulation of cell growth, differentiation, and (mis)function. However, while the functional characterization of short non-coding RNAs has been reaching maturity, there is still a paucity of well characterized long noncoding RNAs, even though large studies in recent years are rapidly increasing the number of annotated ones. The long noncoding RNA PVT1 is encoded by a gene that has been long known since it resides in the well-known cancer risk region 8q24. However, a couple of accidental concurrent conditions have slowed down the study of this gene, that is, a preconception on the primacy of the protein-coding over noncoding RNAs and the prevalent interest in its neighbor MYC oncogene. Recent studies have brought PVT1 under the spotlight suggesting interesting models of functioning, such as competing endogenous RNA activity and regulation of protein stability of important oncogenes, primarily of the MYC oncogene. Despite some advancements in modelling the PVT1 role in cancer, there are many questions that remain unanswered concerning the precise molecular mechanisms underlying its functioning.

microRNA-181a enhances cell proliferation in acute lymphoblastic leukemia by targeting EGR1.

Acute lymphoblastic leukemia (ALL) is an aggressive cancer that occurs in both children and adults. Starting from an integrated analysis of miRNA/mRNA expression profiles in 20 ALL patients, we identify a negative correlation between miR-181a and EGR1. Coherently, miR-181a over-expression in Jurkat T-ALL cells decreases EGR1 expression, increasing cell proliferation and enhancing the cell-cycle progression from G1 to S phase. We show that EGR1 is a new direct target of miR-181a. Our findings suggest that miR-181a behaves as an onco-miRNA in ALL by down-regulating EGR1.

Increased chronic lymphocytic leukemia proliferation upon IgM stimulation is sustained by the upregulation of miR-132 and miR-212.

To assess the involvement of microRNAs (miRNAs) in B-cell receptor (BCR) stimulation, we first evaluated miRNA profiling following IgM cross-linking in chronic lymphocytic leukemia (CLL) cells and in normal B lymphocytes. Second, we combined miRNA and gene expression data to identify putative miRNA functional networks. miRNA profiling showed distinctive patterns of regulation after stimulation in leukemic versus normal B lymphocytes and identified a differential responsiveness to BCR engagement in CLL subgroups according to the immunoglobulin heavy chain variable region mutational status and clinical outcome. The most significantly modulated miRNAs in stimulated CLL are miR-132 and miR-212. Notably, these miRNAs appeared regulated in progressive but not in stable CLL. Accordingly, gene profiling showed a significant transcriptional response to stimulation exclusively in progressive CLL. Based on these findings, we combined miRNA and gene expression data to investigate miR-132 and miR-212 candidate interactions in this CLL subgroup. Correlation analysis pointed to a link between these miRNAs and RB/E2F and TP53 cascades with proproliferative effects, as corroborated by functional analyses. Finally, basal levels of miR-132 and miR-212 were measured in an independent cohort of 20 unstimulated CLL cases and both showed lower expression in progressive compared to stable patients, suggesting an association between the expression of these molecules and disease prognosis. Overall, our results support a model involving miR-132 and miR-212 upregulation in sustaining disease progression in CLL. These miRNAs may therefore provide new valuable strategies for therapeutic intervention.

Arsenic exposure triggers a shift in microRNA expression.

Exposure to inorganic Arsenic (iAs) through drinking water is a major public health problem affecting most countries. iAs has been classified by the International Agency for Research on Cancer as Group 1: "Carcinogenic to humans". Although numerous studies have shown the related adverse effects of iAs, sensitive appropriate biomarkers for studies of environmental epidemiology are still required. The present work aims at investigate the role of microRNAs (miRNAs), powerful negative regulators of gene expression, playing a key role in many physiological and pathological cellular processes, in iAs exposure. To this end, we analyzed miRNA changes in expression profile triggered by iAs exposure in Jurkat cell line. We used microarray technology to profile the expression of miRNAs following 2 µmol/L sodium arsenite treatment at different time points. Moreover, we performed phenotypic analysis of iAs treated cells. Real Time Polymerase Chain Reaction (RT-PCR) was used to validate miRNA microarray data and to assay expression modulation of selected relevant mRNAs. Finally, bioinformatics techniques were applied to reconstruct iAs-relevant molecular pathways and miRNA regulatory networks from the expression data. We report miRNAs modulated after iAs treatment in Jurkat cells. In particular, we highlight 36 miRNAs exhibiting consistent dysregulation and particularly a panel of 8 miRNAs which we also validated by RT-PCR analysis. Computational analysis of lists of putative target genes for these 8 miRNAs points to an involvement in arsenic-response pathways, for a subset of them, that were analyzed by RT-PCR. Furthermore, iAs exposure reveals induction of cell cycle progression and the failure of apoptosis, supporting the idea of iAs carcinogenic activity. Our study provides a list of miRNAs whose expression levels are affected by iAs treatment, corroborating the importance of proceeding with the hunt for specific subset of miRNAs, which can serve as potential biomarkers of iAs effects with useful diagnostic value.

The expression of vasoactive intestinal peptide receptor 1 is negatively modulated by microRNA 525-5p.

BACKGROUND: The human Vasoactive Intestinal Peptide (VIP) is a neurokine with effects on the immune system where it is involved in promoting tolerance. In this context, one of its receptors, VPAC1, has been found to be down-modulated in cells of the immune network in response to activating stimuli. In particular, the bacterial liposaccharide (LPS), a strong activator of the innate immune system, induces a rapid decrease of VPAC1 expression in monocytes and this event correlates with polymorphisms in the 3'-UTR of the gene. METHODOLOGY/PRINCIPAL FINDINGS: MicroRNA 525-5p, having as putative target the 3'-UTR region of VPAC1, has been analysed for its expression in monocytes and for its role in down-modulating VPAC1 expression. We report here that miR-525-5p is promptly up-regulated in LPS-treated monocytes. This microRNA, when co-transfected in 293T cells together with a construct containing the 3'-UTR of the VPAC1 gene, significantly reduced the luciferase activity in a standard expression assay. The U937 cell line as well as primary monocytes enforced to express miR-525-5p, both down-modulate VPAC1 expression at similar extent. CONCLUSIONS/SIGNIFICANCE: Our results show that the response to an inflammatory stimulus elicits in monocytes a rapid increase of miR-525-5p that targets a signaling pathway involved in the control of the immune homeostasis.

An emerging player in the adaptive immune response: microRNA-146a is a modulator of IL-2 expression and activation-induced cell death in T lymphocytes.

Activation of the T cell-mediated immune response has been associated with changes in the expression of specific microRNAs (miRNAs). However, the role of miRNAs in the development of an effective immune response is just beginning to be explored. This study focuses on the functional role of miR-146a in T lymphocyte-mediated immune response and provides interesting clues on the transcriptional regulation of miR-146a during T-cell activation. We show that miR-146a is low in human naive T cells and is abundantly expressed in human memory T cells; consistently, miR-146a is induced in human primary T lymphocytes upon T-cell receptor (TCR) stimulation. Moreover, we identified NF-kB and c-ETS binding sites as required for the induction of miR-146a transcription upon TCR engagement. Our results demonstrate that several signaling pathways, other than inflammation, are influenced by miR-146a. In particular, we provide experimental evidence that miR-146a modulates activation-induced cell death (AICD), acting as an antiapoptotic factor, and that Fas-associated death domain (FADD) is a target of miR-146a. Furthermore, miR-146a enforced expression impairs both activator protein 1 (AP-1) activity and interleukin-2 (IL-2) production induced by TCR engagement, thus suggesting a role of this miRNA in the modulation of adaptive immunity.

Characterization of B- and T-lineage acute lymphoblastic leukemia by integrated analysis of MicroRNA and mRNA expression profiles.

Acute lymphoblastic leukemia (ALL) is an heterogeneous disease comprising several subentities that differ for both immunophenotypic and molecular characteristics. Over the years, the biological understanding of this neoplasm has largely increased. Gene expression profiling has allowed to identify specific signatures for the different ALL subsets and permitted the identification of pathways deregulated by a given lesion. MicroRNAs (miRNAs) are small noncoding RNAs, which play a pivotal role in several cellular functions. In this study, we investigated miRNAs expression profiles in a series of adult ALL cases by microarray analysis. Unsupervised hierarchical clustering largely recapitulated ALL subgroups. Furthermore, we identified miR-148, miR-151, and miR-424 as discriminative of T-lineage versus B-lineage ALL; ANOVA highlighted a set of six miRNAs-namely miR-425-5p, miR-191, miR-146b, miR-128, miR-629, and miR-126-that can discriminate B-lineage ALL subgroups harboring specific molecular lesions. These results were confirmed and extended by quantitative-PCR on a further cohort of cases. Finally, we used Pearson correlation analysis to combine miRNA and gene expression profiles. The distribution of correlation coefficients generated by comparing the expression of every miRNA/gene pair in our data set shows enrichment of both positively and negatively correlated pairs over background distributions obtained using randomized data. Moreover, a clear enrichment for predicted miRNA:target pairs is observed at negative correlation coefficient intervals. Signal-to-noise ratio highlighted several miRNA/gene pairs with a possible role in the disease. In fact, gene set enrichment analysis of genes composing the selected miRNA/gene pairs displays over-representation of functional categories related to cancer and cell-cycle regulation.

Altered microRNA Expression Patterns in Hepatoblastoma Patients.

Liver cancers in children are rare representing only 1.1% of malignancies, with an annual incidence rate of 1.5 cases per million. Hepatoblastoma and hepatocellular carcinomas are the most common malignancies of the liver occurring in young people aged 15 years or younger. Molecular basis of both tumors are still unclear, and common markers (i.e., CTNNB1, APC, IGF-2) are not always useful in the characterization of sporadic forms; in this respect, microRNA recently associated with carcinogenesis could play a pivotal role in their onset. CTNNB1 and APC were analyzed by sequencing, and IGF-2 promoter methylation status was assessed by methylation-specific polymerase chain reaction. MicroRNA expression was assayed by microarray and quantitative reverse transcription-polymerase chain reaction in hepatoblastoma samples. Although few genomic alterations were detected in ours samples, an altered expression of somemicroRNA in hepatoblastoma was observed. Unsupervised clustering shows that microRNA profile can distinguish tumor from nontumor tissues. Further analyses of microRNA contents in hepatoblastoma compared with hepatocellular carcinoma highlighted four upregulated microRNA (miR-214, miR-199a, miR-150 [P < .01], and miR-125a [P < .05]) and one downregulated microRNA (miR-148a [P < .01]). In conclusion, although our samples were poorly informative from a genetic point of view, they showed a peculiar microRNA expression pattern compared with nontumor tissues and hepatocellular carcinoma. MicroRNA could represent valid markers for the classification of pediatric liver tumors.

MicroRNAs: novel regulators of immunity.

MicroRNAs (miRNAs) are a growing class of evolutionarily conserved small non-coding RNAs that act as key regulators of gene expression at post-transcriptional level by targeting mRNAs for translational repression or degradation. These tiny regulators of gene expression have been shown to have unique tissue-specific, developmental stage-specific and disease-specific patterns. These observations suggest that miRNAs might be essential players in cell differentiation and maintenance of tissue type identity. Indeed, during the last years several studies highlighted that miRNAs play a critical role in the differentiation and function of the adaptive and innate immune systems. This review provides an overview of the miRNAs mode of action and of the important and diverse roles of miRNAs in regulating the development of hematopoietic system and in modulating immune responses. The implications arising in the field of autoimmune diseases will be discussed.

A mammalian microRNA expression atlas based on small RNA library sequencing.

MicroRNAs (miRNAs) are small noncoding regulatory RNAs that reduce stability and/or translation of fully or partially sequence-complementary target mRNAs. In order to identify miRNAs and to assess their expression patterns, we sequenced over 250 small RNA libraries from 26 different organ systems and cell types of human and rodents that were enriched in neuronal as well as normal and malignant hematopoietic cells and tissues. We present expression profiles derived from clone count data and provide computational tools for their analysis. Unexpectedly, a relatively small set of miRNAs, many of which are ubiquitously expressed, account for most of the differences in miRNA profiles between cell lineages and tissues. This broad survey also provides detailed and accurate information about mature sequences, precursors, genome locations, maturation processes, inferred transcriptional units, and conservation patterns. We also propose a subclassification scheme for miRNAs for assisting future experimental and computational functional analyses.

Quantitative technologies establish a novel microRNA profile of chronic lymphocytic leukemia.

MicroRNAs (miRNAs) are a novel class of small noncoding RNAs that modulate the expression of genes at the posttranscriptional level. These small molecules have been shown to be involved in cancer, apoptosis, and cell metabolism. In the present study we provide an informative profile of the expression of miRNAs in primary chronic lymphocytic leukemia (CLL) cells using 2 independent and quantitative methods: miRNA cloning and quantitative real-time-polymerase chain reaction (qRT-PCR) of mature miRNAs. Both approaches show that miR-21 and miR-155 are dramatically overexpressed in patients with CLL, although the corresponding genomic loci are not amplified. miR-150 and miR-92 are also significantly deregulated in patients with CLL. In addition, we detected a marked miR-15a and miR-16 decrease in about 11% of cases. Finally, we identified a set of miRNAs whose expression correlates with biologic parameters of prognostic relevance, particularly with the mutational status of the IgV(H) genes. In summary, the results of this study offer for the first time a comprehensive and quantitative profile of miRNA expression in CLL and their healthy counterpart, suggesting that miRNAs could play a primary role in the disease itself.