miR-200 deficiency promotes lung cancer metastasis by activating Notch signaling in cancer-associated fibroblasts.

Lung adenocarcinoma, the most prevalent lung cancer subtype, is characterized by its high propensity to metastasize. Despite the importance of metastasis in lung cancer mortality, its underlying cellular and molecular mechanisms remain largely elusive. Here, we identified miR-200 miRNAs as potent suppressors for lung adenocarcinoma metastasis. miR-200 expression is specifically repressed in mouse metastatic lung adenocarcinomas, and miR-200 decrease strongly correlates with poor patient survival. Consistently, deletion of mir-200c/141 in the KrasLSL-G12D/+ ; Trp53flox/flox lung adenocarcinoma mouse model significantly promoted metastasis, generating a desmoplastic tumor stroma highly reminiscent of metastatic human lung cancer. miR-200 deficiency in lung cancer cells promotes the proliferation and activation of adjacent cancer-associated fibroblasts (CAFs), which in turn elevates the metastatic potential of cancer cells. miR-200 regulates the functional interaction between cancer cells and CAFs, at least in part, by targeting Notch ligand Jagged1 and Jagged2 in cancer cells and inducing Notch activation in adjacent CAFs. Hence, the interaction between cancer cells and CAFs constitutes an essential mechanism to promote metastatic potential.

A genome-wide RNAi screen identifies factors required for distinct stages of C. elegans piRNA biogenesis.

In animals, piRNAs and their associated Piwi proteins guard germ cell genomes against mobile genetic elements via an RNAi-like mechanism. In Caenorhabditis elegans, 21U-RNAs comprise the piRNA class, and these collaborate with 22G RNAs via unclear mechanisms to discriminate self from nonself and selectively and heritably silence the latter. Recent work indicates that 21U-RNAs are post-transcriptional processing products of individual transcription units that produce ∼ 26-nucleotide capped precursors. However, nothing is known of how the expression of precursors is controlled or how primary transcripts give rise to mature small RNAs. We conducted a genome-wide RNAi screen to identify components of the 21U biogenesis machinery. Screening by direct, quantitative PCR (qPCR)-based measurements of mature 21U-RNA levels, we identified 22 genes important for 21U-RNA production, termed TOFUs (Twenty-One-u Fouled Ups). We also identified seven genes that normally repress 21U production. By measuring mature 21U-RNA and precursor levels for the seven strongest hits from the screen, we assigned factors to discrete stages of 21U-RNA production. Our work identifies for the first time factors separately required for the transcription of 21U precursors and the processing of these precursors into mature 21U-RNAs, thereby providing a resource for studying the biogenesis of this important small RNA class.

A genome-wide RNAi screen draws a genetic framework for transposon control and primary piRNA biogenesis in Drosophila.

A large fraction of our genome consists of mobile genetic elements. Governing transposons in germ cells is critically important, and failure to do so compromises genome integrity, leading to sterility. In animals, the piRNA pathway is the key to transposon constraint, yet the precise molecular details of how piRNAs are formed and how the pathway represses mobile elements remain poorly understood. In an effort to identify general requirements for transposon control and components of the piRNA pathway, we carried out a genome-wide RNAi screen in Drosophila ovarian somatic sheet cells. We identified and validated 87 genes necessary for transposon silencing. Among these were several piRNA biogenesis factors. We also found CG3893 (asterix) to be essential for transposon silencing, most likely by contributing to the effector step of transcriptional repression. Asterix loss leads to decreases in H3K9me3 marks on certain transposons but has no effect on piRNA levels.

Probing the initiation and effector phases of the somatic piRNA pathway in Drosophila.

Combining RNAi in cultured cells and analysis of mutant animals, we probed the roles of known Piwi-interacting RNA (piRNA) pathway components in the initiation and effector phases of transposon silencing. Squash associated physically with Piwi, and reductions in its expression led to modest transposon derepression without effects on piRNAs, consistent with an effector role. Alterations in Zucchini or Armitage reduced both Piwi protein and piRNAs, indicating functions in the formation of a stable Piwi RISC (RNA-induced silencing complex). Notably, loss of Zucchini or mutations within its catalytic domain led to accumulation of unprocessed precursor transcripts from flamenco, consistent with a role for this putative nuclease in piRNA biogenesis.

Evaluation of quantitative miRNA expression platforms in the microRNA quality control (miRQC) study.

MicroRNAs are important negative regulators of protein-coding gene expression and have been studied intensively over the past years. Several measurement platforms have been developed to determine relative miRNA abundance in biological samples using different technologies such as small RNA sequencing, reverse transcription-quantitative PCR (RT-qPCR) and (microarray) hybridization. In this study, we systematically compared 12 commercially available platforms for analysis of microRNA expression. We measured an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples and synthetic spikes from microRNA family members with varying homology. We developed robust quality metrics to objectively assess platform performance in terms of reproducibility, sensitivity, accuracy, specificity and concordance of differential expression. The results indicate that each method has its strengths and weaknesses, which help to guide informed selection of a quantitative microRNA gene expression platform for particular study goals.

MicroRNA programs in normal and aberrant stem and progenitor cells.

Emerging evidence suggests that microRNAs (miRNAs), an abundant class of ∼22-nucleotide small regulatory RNAs, play key roles in controlling the post-transcriptional genetic programs in stem and progenitor cells. Here we systematically examined miRNA expression profiles in various adult tissue-specific stem cells and their differentiated counterparts. These analyses revealed miRNA programs that are common or unique to blood, muscle, and neural stem cell populations and miRNA signatures that mark the transitions from self-renewing and quiescent stem cells to proliferative and differentiating progenitor cells. Moreover, we identified a stem/progenitor transition miRNA (SPT-miRNA) signature that predicts the effects of genetic perturbations, such as loss of PTEN and the Rb family, AML1-ETO9a expression, and MLL-AF10 transformation, on self-renewal and proliferation potentials of mutant stem/progenitor cells. We showed that some of the SPT-miRNAs control the self-renewal of embryonic stem cells and the reconstitution potential of hematopoietic stem cells (HSCs). Finally, we demonstrated that SPT-miRNAs coordinately regulate genes that are known to play roles in controlling HSC self-renewal, such as Hoxb6 and Hoxa4. Together, these analyses reveal the miRNA programs that may control key processes in normal and aberrant stem and progenitor cells, setting the foundations for dissecting post-transcriptional regulatory networks in stem cells.

MicroRNA regulation of molecular networks mapped by global microRNA, mRNA, and protein expression in activated T lymphocytes.

MicroRNAs (miRNAs) regulate specific immune mechanisms, but their genome-wide regulation of T lymphocyte activation is largely unknown. We performed a multidimensional functional genomics analysis to integrate genome-wide differential mRNA, miRNA, and protein expression as a function of human T lymphocyte activation and time. We surveyed expression of 420 human miRNAs in parallel with genome-wide mRNA expression. We identified a unique signature of 71 differentially expressed miRNAs, 57 of which were previously not known as regulators of immune activation. The majority of miRNAs are upregulated, mRNA expression of these target genes is downregulated, and this is a function of binding multiple miRNAs (combinatorial targeting). Our data reveal that consideration of this complex signature, rather than single miRNAs, is necessary to construct a full picture of miRNA-mediated regulation. Molecular network mapping of miRNA targets revealed the regulation of activation-induced immune signaling. In contrast, pathways populated by genes that are not miRNA targets are enriched for metabolism and biosynthesis. Finally, we specifically validated miR-155 (known) and miR-221 (novel in T lymphocytes) using locked nucleic acid inhibitors. Inhibition of these two highly upregulated miRNAs in CD4(+) T cells was shown to increase proliferation by removing suppression of four target genes linked to proliferation and survival. Thus, multiple lines of evidence link top functional networks directly to T lymphocyte immunity, underlining the value of mapping global gene, protein, and miRNA expression.

A microRNA component of the p53 tumour suppressor network.

A global decrease in microRNA (miRNA) levels is often observed in human cancers, indicating that small RNAs may have an intrinsic function in tumour suppression. To identify miRNA components of tumour suppressor pathways, we compared miRNA expression profiles of wild-type and p53-deficient cells. Here we describe a family of miRNAs, miR-34a-c, whose expression reflected p53 status. Genes encoding miRNAs in the miR-34 family are direct transcriptional targets of p53, whose induction by DNA damage and oncogenic stress depends on p53 both in vitro and in vivo. Ectopic expression of miR-34 induces cell cycle arrest in both primary and tumour-derived cell lines, which is consistent with the observed ability of miR-34 to downregulate a programme of genes promoting cell cycle progression. The p53 network suppresses tumour formation through the coordinated activation of multiple transcriptional targets, and miR-34 may act in concert with other effectors to inhibit inappropriate cell proliferation.

The microRNA body map: dissecting microRNA function through integrative genomics.

While a growing body of evidence implicates regulatory miRNA modules in various aspects of human disease and development, insights into specific miRNA function remain limited. Here, we present an innovative approach to elucidate tissue-specific miRNA functions that goes beyond miRNA target prediction and expression correlation. This approach is based on a multi-level integration of corresponding miRNA and mRNA gene expression levels, miRNA target prediction, transcription factor target prediction and mechanistic models of gene network regulation. Predicted miRNA functions were either validated experimentally or compared to published data. The predicted miRNA functions are accessible in the miRNA bodymap, an interactive online compendium and mining tool of high-dimensional newly generated and published miRNA expression profiles. The miRNA bodymap enables prioritization of candidate miRNAs based on their expression pattern or functional annotation across tissue or disease subgroup. The miRNA bodymap project provides users with a single one-stop data-mining solution and has great potential to become a community resource.

Development and clinical applications of an enclosed automated targeted NGS library preparation system.

The rapid development of next-generation sequencing (NGS) technology has promoted its wide clinical application in precision medicine for oncology. However, laborious and time-consuming manual operations, highly skilled personnel requirements, and cross-contamination are major challenges for the clinical implementation of NGS technology-based tests. The Automated NGS Diagnostic Solutions (ANDiS) 500 system is a fully enclosed cassette-dependent automated NGS library preparation system. This platform could produce qualified targeted amplicon library in three steps with only 15 min of hands-on time. Rigorous cross-contamination test using simulated contaminant plasmids confirmed that the design of disposable cassette guarantees zero sample cross-contamination. The BRCA1 and BRCA2 mutation detection panel and gastrointestinal cancer-related gene analysis panel for the ANDiS 500 platform showed 100% accuracy and precision in detecting germ-line mutations and somatic mutations respectively. Furthermore, those panels showed 100% concordance with verified methods in a prospective cohort study enrolling 363 patients and a cohort of 45 pan-cancer samples. In conclusion, the ANDiS 500 automated platform could overcome major challenges for implementing NGS assays clinically and is eligible for routine clinical tests.

Comprehensive Evaluation and Application of a Novel Method to Isolate Cell-Free DNA Derived From Bile of Biliary Tract Cancer Patients.

Cell-free DNA (cfDNA) exists in various types of bodily fluids, including plasma, urine, bile, and others. Bile cfDNA could serve as a promising liquid biopsy for biliary tract cancer (BTC) patients, as bile directly contacts tumors in the biliary tract system. However, there is no commercial kit or widely acknowledged method for bile cfDNA extraction. In this study, we established a silica-membrane-based method, namely 3D-BCF, for bile cfDNA isolation, exhibiting effective recovery of DNA fragments in the spike-in assay. We then compared the 3D-BCF method with four other commercial kits: the BIOG cfDNA Easy Kit (BIOG), QIAamp DNA Mini Kit (Qiagen), MagMAXTM Cell-Free DNA Isolation Kit (Thermo Fisher), and NORGEN Urine Cell-Free Circulating DNA Purification Mini Kit (Norgen Biotek). The proposed 3D-BCF method exhibited the highest cfDNA isolation efficiency (p < 0.0001) from patient bile samples, and bile cfDNA of short, medium or long fragments could all be extracted effectively. To test whether the extracted bile cfDNA from patients carries tumor-related genomic information, we performed next-generation sequencing on the cfDNA and verified the gene-mutation results by polymerase chain reaction (PCR)-Sanger chromatograms and copy-number-variation (CNV) detection by fluorescence in situ hybridization (FISH) of tumor tissues. The 3D-BCF method could efficiently extract cfDNA from bile samples, providing technical support for bile cfDNA as a promising liquid biopsy for BTC patient diagnosis and prognosis.

MicroRNA characterize genetic diversity and drug resistance in pediatric acute lymphoblastic leukemia.

BACKGROUND: MicroRNA regulate the activity of protein-coding genes including those involved in hematopoietic cancers. The aim of the current study was to explore which microRNA are unique for seven different subtypes of pediatric acute lymphoblastic leukemia. DESIGN AND METHODS: Expression levels of 397 microRNA (including novel microRNA) were measured by quantitative real-time polymerase chain reaction in 81 cases of pediatric leukemia and 17 normal hematopoietic control cases. RESULTS: All major subtypes of acute lymphoblastic leukemia, i.e. T-cell, MLL-rearranged, TEL-AML1-positive, E2A-PBX1-positive and hyperdiploid acute lymphoblastic leukemia, with the exception of BCR-ABL-positive and 'B-other' acute lymphoblastic leukemias (defined as precursor B-cell acute lymphoblastic leukemia not carrying the foregoing cytogenetic aberrations), were found to have unique microRNA-signatures that differed from each other and from those of healthy hematopoietic cells. Strikingly, the microRNA signature of TEL-AML1-positive and hyperdiploid cases partly overlapped, which may suggest a common underlying biology. Moreover, aberrant down-regulation of let-7b (~70-fold) in MLL-rearranged acute lymphoblastic leukemia was linked to up-regulation of oncoprotein c-Myc (P(FDR)<0.0001). Resistance to vincristine and daunorubicin was characterized by an approximately 20-fold up-regulation of miR-125b, miR-99a and miR-100 (P(FDR)≤0.002). No discriminative microRNA were found for prednisolone response and only one microRNA was linked to resistance to L-asparaginase. A combined expression profile based on 14 microRNA that were individually associated with prognosis, was highly predictive of clinical outcome in pediatric acute lymphoblastic leukemia (5-year disease-free survival of 89.4%±7% versus 60.8±12%, P=0.001). CONCLUSIONS: Genetic subtypes and drug-resistant leukemic cells display characteristic microRNA signatures in pediatric acute lymphoblastic leukemia. Functional studies of discriminative and prognostically important microRNA may provide new insights into the biology of pediatric acute lymphoblastic leukemia.

Development and Validation of an RNA Sequencing Assay for Gene Fusion Detection in Formalin-Fixed, Paraffin-Embedded Tumors.

RNA sequencing (RNA-seq) is a well-validated tool for detecting gene fusions in fresh-frozen tumors; however, RNA-seq is much more challenging to use with formalin-fixed, paraffin-embedded (FFPE) tumor samples. We evaluated the performance of RNA-seq to detect gene fusions in clinical FFPE tumor samples. Our assay identified all 15 spiked-in NTRK fusions from RNA reference material and six known fusions from five cancer cell lines. Limit of detection for the assay was assessed with a series of dilutions of RNA from the cell line H2228. These fusions can be detected when the dilution is down to 10%. Good intra-assay and interassay reproducibility was observed in three specimens. For clinical validation, the assay detected 10 of 12 fusions initially identified by a DNA panel (covering 23 gene fusions) in clinical specimens (83.3% sensitivity), whereas one fusion (MET fusion) was identified in another 34 fusion-negative tumor specimens as determined by the DNA panel (negative prediction value of 94.3%). This MET fusion was confirmed by RT-PCR and Sanger sequencing, which found that this is a false-negative result for the DNA panel. The assay also identified 26 extra fusions not covered by the DNA panel, 20 (76.9%) of which were validated by RT-PCR and Sanger sequencing. Therefore, this RNA assay has reasonable performance and could complement DNA-based next-generation sequencing assays.

High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA.

MicroRNAs (miRNAs) are an emerging class of small non-coding RNAs implicated in a wide variety of cellular processes. Research in this field is accelerating, and the growing number of miRNAs emphasizes the need for high-throughput and sensitive detection methods. Here we present the successful evaluation of the Megaplex reverse transcription format of the stem-loop primer-based real-time quantitative polymerase chain reaction (RT-qPCR) approach to quantify miRNA expression. The Megaplex reaction provides simultaneous reverse transcription of 450 mature miRNAs, ensuring high-throughput detection. Further, the introduction of a complementary DNA pre-amplification step significantly reduces the amount of input RNA needed, even down to single-cell level. To evaluate possible pre-amplification bias, we compared the expression of 384 miRNAs in three different cancer cell lines with Megaplex RT, with or without an additional pre-amplification step. The normalized Cq values of all three sample pairs showed a good correlation with maintenance of differential miRNA expression between the cell lines. Moreover, pre-amplification using 10 ng of input RNA enabled the detection of miRNAs that were undetectable when using Megaplex alone with 400 ng of input RNA. The high specificity of RT-qPCR together with a superior sensitivity makes this approach the method of choice for high-throughput miRNA expression profiling.

MicroRNAs in embryonic stem cells.

The unique properties of embryonic stem cells (ESCs) to self-renew indefinitely or to differentiate to any cell type have great potential for clinical applications in regenerative medicine. MicroRNAs (miRNAs) are emerging as important regulators of post-transcriptional gene expression and have been implicated as crucial elements in regulating ESCs. Here, we review recent progresses in characterizing the role of miRNAs in the maintenance and development of ESCs.

Gene expression in stem cells.

Both embryonic and adult stem cells promise a precious cell source for the treatment of various degenerative diseases due to their capacity for self-renewal and ability to differentiate along multiple cell lineages. A lack of understanding surrounding the basic biology of stem cells, the stemness, continues to challenge successful clinical application of these cells. Gene expression analysis, both genome-wide and targeted at specific gene subsets, has played a key role in improving our understanding of the genetic attributes of stem cells through identification of molecular signatures that characterize normal stem cell function. Such approaches have facilitated the investigation of stemness in a subset of tumor cells, so-called cancer stem cells, and have started to unravel their roles in initiation and progression of tumors. This review summarizes recent advances of gene expression analysis in stem cell research and also discusses critical issues that remain to be elucidated in the search for cancer stem cells markers.

Characterization of microRNA expression levels and their biological correlates in human cancer cell lines.

MicroRNAs are small noncoding RNAs that function by regulating target gene expression posttranscriptionally. They play a critical role in developmental and physiologic processes and are implicated in the pathogenesis of several human diseases including cancer. We examined the expression profiles of 241 human microRNAs in normal tissues and the NCI-60 panel of human tumor-derived cell lines. To quantify microRNA expression, we employed a highly sensitive technique that uses stem-loop primers for reverse transcription followed by real-time PCR. Most microRNAs were expressed at lower levels in tumor-derived cell lines compared with the corresponding normal tissue. Agglomerative hierarchical clustering analysis of microRNA expression revealed four groups among the NCI-60 cell lines consisting of hematologic, colon, central nervous system, and melanoma tumor-derived cell lines clustered in a manner that reflected their tissue of origin. We identified specific subsets of microRNAs that provide candidate molecular signatures characteristic of the tumor-derived cell lines belonging to these four clusters. We also identified specific microRNA expression patterns that correlated with the proliferation indices of the NCI-60 cell lines, and we developed evidence for the identification of specific microRNAs as candidate oncogenes and tumor suppressor genes in different tumor types. Our results provide evidence that microRNA expression patterns may mark specific biological characteristics of tumors and/or mediate biological activities important for the pathobiology of malignant tumors. These findings call attention to the potential of microRNAs to provide etiologic insights as well as to serve as both diagnostic markers and therapeutic targets for many different tumor types.

Comparison of TaqMan, KASP and rhAmp SNP genotyping platforms in hexaploid wheat.

Advances in high-throughput genotyping enable the generation of genome-scale data much more easily and at lower cost than ever before. However, small-scale and cost-effective high-throughput single-nucleotide polymorphism (SNP) genotyping technologies are still under development. In this study, we compared the performances of TaqMan, KASP and rhAmp SNP genotyping platforms in terms of their assay design flexibility, assay design success rate, allele call rate and quality, ease of experiment run and cost per sample. Fifty SNP markers linked to genes governing various agronomic traits of wheat were chosen to design SNP assays. Design success rates were 39/50, 49/50, and 49/50 for TaqMan, KASP, and rhAmp, respectively, and 30 SNP assays were manufactured for genotyping comparisons across the three platforms. rhAmp showed 97% of samples amplified while TaqMan and KASP showed 93% and 93.5% of amplifications, respectively. Allele call quality of rhAmp was 97%, while it was 98% for both TaqMan and KASP. rhAmp and KASP showed significantly better (p < 0.001) allele discrimination than TaqMan; however, TaqMan showed the most compact cluster. Based on the current market, rhAmp was the least expensive technology followed by KASP. In conclusion, rhAmp provides a reliable and cost-effective option for targeted genotyping and marker-assisted selection in crop genetic improvement.

Identification of miRNA changes in Alzheimer's disease brain and CSF yields putative biomarkers and insights into disease pathways.

MicroRNAs have essential functional roles in brain development and neuronal specification but their roles in neurodegenerative diseases such as Alzheimer's disease (AD) is unknown. Using a sensitive qRT-PCR platform we identified regional and stage-specific deregulation of miRNA expression in AD patient brains. We used experimental validation in addition to literature to reveal how the deregulated brain microRNAs are biomarkers for known and novel pathways in AD pathogenesis related to amyloid processing, neurogenesis, insulin resistance, and innate immunity. We additionally recovered miRNAs from cerebrospinal fluid and discovered AD-specific miRNA changes consistent with their role as potential biomarkers of disease.

Characterization of microRNA expression profiles in normal human tissues.

BACKGROUND: Measuring the quantity of miRNAs in tissues of different physiological and pathological conditions is an important first step to investigate the functions of miRNAs. Matched samples from normal state can provide essential baseline references to analyze the variation of miRNA abundance. RESULTS: We provided expression data of 345 miRNAs in 40 normal human tissues, which identified universally expressed miRNAs, and several groups of miRNAs expressed exclusively or preferentially in certain tissue types. Many miRNAs with co-regulated expression patterns are located within the same genomic clusters, and candidate transcriptional factors that control the pattern of their expression may be identified by a comparative genomic strategy. Hierarchical clustering of normal tissues by their miRNA expression profiles basically followed the structure, anatomical locations, and physiological functions of the organs, suggesting that functions of a miRNA could be appreciated by linking to the biologies of the tissues in which it is uniquely expressed. Many predicted target genes of miRNAs that had specific reduced expression in brain and peripheral blood mononuclear cells are required for embryonic development of the nervous and hematopoietic systems based on database search. CONCLUSION: We presented a global view of tissue distribution of miRNAs in relation to their chromosomal locations and genomic structures. We also described evidence from the cis-regulatory elements and the predicted target genes of miRNAs to support their tissue-specific functional roles to regulate the physiologies of the normal tissues in which they are expressed.