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1.  A shared transcriptional program in early breast neoplasias despite genetic and clinical distinctions 
Genome Biology  2014;15(5):R71.
Background
The earliest recognizable stages of breast neoplasia are lesions that represent a heterogeneous collection of epithelial proliferations currently classified based on morphology. Their role in the development of breast cancer is not well understood but insight into the critical events at this early stage will improve efforts in breast cancer detection and prevention. These microscopic lesions are technically difficult to study so very little is known about their molecular alterations.
Results
To characterize the transcriptional changes of early breast neoplasia, we sequenced 3′- end enriched RNAseq libraries from formalin-fixed paraffin-embedded tissue of early neoplasia samples and matched normal breast and carcinoma samples from 25 patients. We find that gene expression patterns within early neoplasias are distinct from both normal and breast cancer patterns and identify a pattern of pro-oncogenic changes, including elevated transcription of ERBB2, FOXA1, and GATA3 at this early stage. We validate these findings on a second independent gene expression profile data set generated by whole transcriptome sequencing. Measurements of protein expression by immunohistochemistry on an independent set of early neoplasias confirms that ER pathway regulators FOXA1 and GATA3, as well as ER itself, are consistently upregulated at this early stage. The early neoplasia samples also demonstrate coordinated changes in long non-coding RNA expression and microenvironment stromal gene expression patterns.
Conclusions
This study is the first examination of global gene expression in early breast neoplasia, and the genes identified here represent candidate participants in the earliest molecular events in the development of breast cancer.
doi:10.1186/gb-2014-15-5-r71
PMCID: PMC4072957  PMID: 24887547
2.  Next generation sequencing-based expression profiling identifies signatures from benign stromal proliferations that define stromal components of breast cancer 
Breast Cancer Research : BCR  2013;15(6):R117.
Introduction
Multiple studies have shown that the tumor microenvironment (TME) of carcinomas can play an important role in the initiation, progression, and metastasis of cancer. Here we test the hypothesis that specific benign fibrous soft tissue tumor gene expression profiles may represent distinct stromal fibroblastic reaction types that occur in different breast cancers. The discovered stromal profiles could classify breast cancer based on the type of stromal reaction patterns in the TME.
Methods
Next generation sequencing-based gene expression profiling (3SEQ) was performed on formalin fixed, paraffin embedded (FFPE) samples of 10 types of fibrous soft tissue tumors. We determined the extent to which these signatures could identify distinct subsets of breast cancers in four publicly available breast cancer datasets.
Results
A total of 53 fibrous tumors were sequenced by 3SEQ with an average of 29 million reads per sample. Both the gene signatures derived from elastofibroma (EF) and fibroma of tendon sheath (FOTS) demonstrated robust outcome results for survival in the four breast cancer datasets. The breast cancers positive for the EF signature (20-33% of the cohort) demonstrated significantly better outcome for survival. In contrast, the FOTS signature-positive breast cancers (11-35% of the cohort) had a worse outcome.
Conclusions
We defined and validated two new stromal signatures in breast cancer (EF and FOTS), which are significantly associated with prognosis. Our group has previously identified novel cancer stromal gene expression signatures associated with outcome differences in breast cancer by gene expression profiling of three soft tissue tumors, desmoid-type fibromatosis (DTF), solitary fibrous tumor (SFT), and tenosynovial giant cell tumor (TGCT/CSF1), as surrogates for stromal expression patterns. By combining the stromal signatures of EF and FOTS, with our previously identified DTF and TGCT/CSF1 signatures we can now characterize clinically relevant stromal expression profiles in the TME for between 74% to 90% of all breast cancers.
doi:10.1186/bcr3586
PMCID: PMC3978842  PMID: 24342436
3.  Recipe for a Busy Bee: MicroRNAs in Honey Bee Caste Determination 
PLoS ONE  2013;8(12):e81661.
Social caste determination in the honey bee is assumed to be determined by the dietary status of the young larvae and translated into physiological and epigenetic changes through nutrient-sensing pathways. We have employed Illumina/Solexa sequencing to examine the small RNA content in the bee larval food, and show that worker jelly is enriched in miRNA complexity and abundance relative to royal jelly. The miRNA levels in worker jelly were 7–215 fold higher than in royal jelly, and both jellies showed dynamic changes in miRNA content during the 4th to 6th day of larval development. Adding specific miRNAs to royal jelly elicited significant changes in queen larval mRNA expression and morphological characters of the emerging adult queen bee. We propose that miRNAs in the nurse bee secretions constitute an additional element in the regulatory control of caste determination in the honey bee.
doi:10.1371/journal.pone.0081661
PMCID: PMC3862878  PMID: 24349106
4.  Cyclin D1 as a diagnostic immunomarker for endometrial stromal sarcoma with YWHAE-FAM22 rearrangement 
Endometrial stromal sarcoma (ESS) characterized by YWHAE-FAM22 genetic fusion is histologically higher-grade and clinically more aggressive than ESS with JAZF1-SUZ12 or equivalent genetic rearrangements, hence it is clinically important to recognize this subset of ESS. To identify diagnostic immunomarkers for this biologically-defined ESS subset, we compared gene expression profiles from YWHAE-FAM22 ESS, JAZF1-rearranged ESS and uterine leiomyosarcomas. These studies showed consistent upregulation of cyclin D1 in YWHAE-FAM22 ESS compared to JAZF1-SUZ12 ESS. Immunohistochemically, the high-grade round cell component of all 12 YWHAE-FAM22 ESS demonstrated diffuse (≥70%) moderate-to-strong nuclear cyclin D1 staining and this diffuse positivity was not seen in 34 ESS with JAZF1 and equivalent genetic rearrangements or in 21 low-grade ESS with no demonstrable genetic rearrangements. In a series of 243 non-ESS pure uterine mesenchymal and mixed epithelial-mesenchymal tumors, only 2 of 8 undifferentiated endometrial sarcomas with nuclear uniformity and 1 of 80 uterine leiomyosarcomas demonstrate diffuse cyclin D1 immunoreactivity. Both cyclin D1-positive undifferentiated endometrial sarcomas showed diffuse strong CD10 staining, which is consistently absent in the high-grade round cell component of YWHAE-FAM22 ESS. The low-grade spindle cell component of YWHAE-FAM22 ESS showed a spatially heterogeneous cyclin D1 staining pattern that was weaker and less diffuse overall. Our findings indicate that cyclin D1 is a sensitive and specific diagnostic immunomarker for YWHAE-FAM22 ESS. When evaluating high-grade uterine sarcomas, cyclin D1 can be included in the immunohistochemical panel as an indicator of YWHAE-FAM22 ESS.
doi:10.1097/PAS.0b013e31825fa931
PMCID: PMC3444748  PMID: 22982899
Endometrial stromal sarcoma; round cell; YWHAE-FAM22; cyclin D1; JAZF1-SUZ12
5.  The novel long non-coding RNA CRG regulates Drosophila locomotor behavior 
Nucleic Acids Research  2012;40(22):11714-11727.
Long non-coding RNAs (lncRNAs) that have no protein-coding capacity make up a large proportion of the transcriptome of various species. Many lncRNAs are expressed within the animal central nervous system in spatial- and temporal-specific patterns, indicating that lncRNAs play important roles in cellular processes, neural development, and even in cognitive and behavioral processes. However, relatively little is known about their in vivo functions and underlying molecular mechanisms in the nervous system. Here, we report a neural-specific Drosophila lncRNA, CASK regulatory gene (CRG), which participates in locomotor activity and climbing ability by positively regulating its neighboring gene CASK (Ca2+/calmodulin-dependent protein kinase). CRG deficiency led to reduced locomotor activity and a defective climbing ability—phenotypes that are often seen in CASK mutant. CRG mutant also showed reduced CASK expression level while CASK over-expression could rescue the CRG mutant phenotypes in reciprocal. At the molecular level, CRG was required for the recruitment of RNA polymerase II to the CASK promoter regions, which in turn enhanced CASK expression. Our work has revealed new functional roles of lncRNAs and has provided insights to explore the pathogenesis of neurological diseases associated with movement disorders.
doi:10.1093/nar/gks943
PMCID: PMC3526303  PMID: 23074190
6.  Transcriptional profiling of long non-coding RNAs and novel transcribed regions across a diverse panel of archived human cancers 
Genome Biology  2012;13(8):R75.
Background
Molecular characterization of tumors has been critical for identifying important genes in cancer biology and for improving tumor classification and diagnosis. Long non-coding RNAs, as a new, relatively unstudied class of transcripts, provide a rich opportunity to identify both functional drivers and cancer-type-specific biomarkers. However, despite the potential importance of long non-coding RNAs to the cancer field, no comprehensive survey of long non-coding RNA expression across various cancers has been reported.
Results
We performed a sequencing-based transcriptional survey of both known long non-coding RNAs and novel intergenic transcripts across a panel of 64 archival tumor samples comprising 17 diagnostic subtypes of adenocarcinomas, squamous cell carcinomas and sarcomas. We identified hundreds of transcripts from among the known 1,065 long non-coding RNAs surveyed that showed variability in transcript levels between the tumor types and are therefore potential biomarker candidates. We discovered 1,071 novel intergenic transcribed regions and demonstrate that these show similar patterns of variability between tumor types. We found that many of these differentially expressed cancer transcripts are also expressed in normal tissues. One such novel transcript specifically expressed in breast tissue was further evaluated using RNA in situ hybridization on a panel of breast tumors. It was shown to correlate with low tumor grade and estrogen receptor expression, thereby representing a potentially important new breast cancer biomarker.
Conclusions
This study provides the first large survey of long non-coding RNA expression within a panel of solid cancers and also identifies a number of novel transcribed regions differentially expressed across distinct cancer types that represent candidate biomarkers for future research.
doi:10.1186/gb-2012-13-8-r75
PMCID: PMC4053743  PMID: 22929540
3SEQ; FFPE; human cancer; intergenic transcripts; lncRNAs; novel transcripts; solid tumors; transcriptional profiling
7.  Identification and characterization of small non-coding RNAs from Chinese fir by high throughput sequencing 
BMC Plant Biology  2012;12:146.
Background
Small non-coding RNAs (sRNAs) play key roles in plant development, growth and responses to biotic and abiotic stresses. At least four classes of sRNAs have been well characterized in plants, including repeat-associated siRNAs (rasiRNAs), microRNAs (miRNAs), trans-acting siRNAs (tasiRNAs) and natural antisense transcript-derived siRNAs. Chinese fir (Cunninghamia lanceolata) is one of the most important coniferous evergreen tree species in China. No sRNA from Chinese fir has been described to date.
Results
To obtain sRNAs in Chinese fir, we sequenced a sRNA library generated from seeds, seedlings, leaves, stems and calli, using Illumina high throughput sequencing technology. A comprehensive set of sRNAs were acquired, including conserved and novel miRNAs, rasiRNAs and tasiRNAs. With BLASTN and MIREAP we identified a total of 115 conserved miRNAs comprising 40 miRNA families and one novel miRNA with precursor sequence. The expressions of 16 conserved and one novel miRNAs and one tasiRNA were detected by RT-PCR. Utilizing real time RT-PCR, we revealed that four conserved and one novel miRNAs displayed developmental stage-specific expression patterns in Chinese fir. In addition, 209 unigenes were predicted to be targets of 30 Chinese fir miRNA families, of which five target genes were experimentally verified by 5' RACE, including a squamosa promoter-binding protein gene, a pentatricopeptide (PPR) repeat-containing protein gene, a BolA-like family protein gene, AGO1 and a gene of unknown function. We also demonstrated that the DCL3-dependent rasiRNA biogenesis pathway, which had been considered absent in conifers, existed in Chinese fir. Furthermore, the miR390-TAS3-ARF regulatory pathway was elucidated.
Conclusions
We unveiled a complex population of sRNAs in Chinese fir through high throughput sequencing. This provides an insight into the composition and function of sRNAs in Chinese fir and sheds new light on land plant sRNA evolution.
doi:10.1186/1471-2229-12-146
PMCID: PMC3462689  PMID: 22894611
Chinese fir; miRNA; rasiRNA; tasiRNA; Cunninghamia lanceolata
8.  Experimental RNomics and genomic comparative analysis reveal a large group of species-specific small non-message RNAs in the silkworm Bombyx mori 
Nucleic Acids Research  2011;39(9):3792-3805.
Accumulating evidences show that small non-protein coding RNAs (ncRNAs) play important roles in development, stress response and other cellular processes. The silkworm is an important model for studies on insect genetics and control of lepidopterous pests. Here, we have performed the first systematic identification and analysis of intermediate size ncRNAs (50–500 nt) in the silkworm. We identified 189 novel ncRNAs, including 141 snoRNAs, six snRNAs, three tRNAs, one SRP and 38 unclassified ncRNAs. Forty ncRNAs showed significantly altered expression during silkworm development or across specific stage transitions. Genomic comparisons revealed that 123 of these ncRNAs are potentially silkworm-specific. Analysis of the genomic organization of the ncRNA loci showed that 32.62% of the novel snoRNA loci are intergenic, and that all the intronic snoRNAs follow the pattern of one-snoRNA-per-intron. Target site analysis predicted a total of 95 2′-O-methylation and pseudouridylation modification sites of rRNAs, snRNAs and tRNAs. Together, these findings provide new clues for future functional study of ncRNA during insect development and evolution.
doi:10.1093/nar/gkq1317
PMCID: PMC3089462  PMID: 21227919
9.  Genome-scale identification of Caenorhabditis elegans regulatory elements by tiling-array mapping of DNase I hypersensitive sites 
BMC Genomics  2009;10:92.
Background
A major goal of post-genomics research is the integrated analysis of genes, regulatory elements and the chromatin architecture on a genome-wide scale. Mapping DNase I hypersensitive sites within the nuclear chromatin is a powerful and well-established method of identifying regulatory element candidates.
Results
Here, we report the first genome-wide analysis of DNase I hypersensitive sites (DHSs) in Caenorhabditis elegans. The data was obtained by hybridizing DNase I-treated and end-captured material from young adult worms to a high-resolution tiling microarray. The data show that C. elegans DHSs were significantly enriched within intergenic regions located 2 kb upstream and downstream of coding genes, and also that a considerable fraction of all DHSs mapped to intergenic positions distant to annotated coding genes. Annotated transcribed loci were generally depleted in DHSs relative to intergenic regions, but DHSs were nonetheless enriched in coding exons and UTRs, whereas introns were significantly depleted in DHSs. Many DHSs appeared to be associated with annotated non-coding RNAs and recently detected transcripts of unknown function. It has been reported that nematode highly conserved non-coding elements were associated with cis-regulatory elements, and we also found that DHSs, particularly distal intergenic DHSs, were significantly enriched in regions that were conserved between the C. elegans and C. briggsae genomes.
Conclusion
We describe the first genome-wide analysis of C. elegans DHSs, and show that the distribution of DHSs is strongly associated with functional elements in the genome.
doi:10.1186/1471-2164-10-92
PMCID: PMC2651899  PMID: 19243610
10.  Transcriptional inhibiton of Hoxd4 expression by miRNA-10a in human breast cancer cells 
BMC Molecular Biology  2009;10:12.
Background
Small noncoding RNAs (ncRNAs), including short interfering RNAs (siRNAs) and microRNAs (miRNAs), can silence genes at the transcriptional, post-transcriptional or translational level [1,2].
Results
Here, we show that microRNA-10a (miR-10a) targets a homologous DNA region in the promoter region of the hoxd4 gene and represses its expression at the transcriptional level. Mutational analysis of the miR-10a sequence revealed that the 3' end of the miRNA sequence is the most critical element for the silencing effect. MicroRNA-10a-induced transcriptional gene inhibition requires the presence of Dicer and Argonautes 1 and 3, and it is related to promoter associated noncoding RNAs. Bisulfite sequencing analysis showed that the reduced hoxd4 expression was accompanied by de novo DNA methylation at the hoxd4 promoter. We further demonstrated that trimethylation of histone 3 lysine 27 (H3K27me3) is involved in the miR-10a-induced hoxd4 transcriptional gene silence.
Conclusion
In conclusion, our results demonstrate that miR-10a can regulate human gene expression in a transcriptional manner, and indicate that endogenous small noncoding RNA-induced control of transcription may be a potential system for expressional regulation in human breast cancer cells.
doi:10.1186/1471-2199-10-12
PMCID: PMC2680403  PMID: 19232136

Results 1-10 (10)