Project description:Purpose: The goal of this study is to compare endothelial small RNA transcriptome to identify the target of OASL under basal or stimulated conditions by utilizing miRNA-seq. Methods: Endothelial miRNA profilies of siCTL or siOASL transfected HUVECs were generated by illumina sequencing method, in duplicate. After sequencing, the raw sequence reads are filtered based on quality. The adapter sequences are also trimmed off the raw sequence reads. rRNA removed reads are sequentially aligned to reference genome (GRCh38) and miRNA prediction is performed by miRDeep2. Results: We identified known miRNA in species (miRDeep2) in the HUVECs transfected with siCTL or siOASL. The expression profile of mature miRNA is used to analyze differentially expressed miRNA(DE miRNA). Conclusions: Our study represents the first analysis of endothelial miRNA profiles affected by OASL knockdown with biologic replicates.
Project description:A cDNA library was constructed by Novogene (CA, USA) using a Small RNA Sample Pre Kit, and Illumina sequencing was conducted according to company workflow, using 20 million reads. Raw data were filtered for quality as determined by reads with a quality score > 5, reads containing N < 10%, no 5' primer contaminants, and reads with a 3' primer and insert tag. The 3' primer sequence was trimmed and reads with a poly A/T/G/C were removed
Project description:YY1 is a sequence-specific DNA-binding transcription factor that has many important biological roles. However, its function in trophoblasts at the maternal-foetal interface remains to be elucidated. In this study, we used an mRNA microarray and quantitative reverse transcription-PCR and compared the YY1 mRNA expression level in trophoblasts between patients with recurrent miscarriage (RM) and healthy control subjects. Our results revealed that YY1 mRNA expression was significantly lower in the trophoblasts of the RM group compared with the healthy control group. Furthermore, immunofluorescence and immunohistochemical data showed that YY1 was highly expressed in human placental villi during early pregnancy, especially in cytotrophoblast cells and invasive extravillous trophoblasts, and it was expressed at a much lower level in the placental villi of term pregnancy. YY1 overexpression enhanced the invasion and proliferation of trophoblasts, while knockdown of YY1 repressed these effects. Antibody array screening revealed that YY1 significantly promoted MMP2 expression in trophoblasts. Bioinformatics analysis identified three YY1-binding sites in the MMP2 promoter region, and chromatin immunoprecipitation analysis verified that YY1 binds directly to its promoter region. Importantly, inhibition of YY1 by siRNA clearly decreased trophoblast invasion in an ex vivo explant culture model. Overall, our findings revealed a new regulatory pathway of YY1/MMP2 in trophoblast cells invasion during early pregnancy, and indicated that YY1 may be involved in the pathogenesis of RM. Total RNA was isolated using Trizol from trophoblast cells from three healthy controls (HC) and three recurrent miscarriage (RM) patients. Total RNA were extracted and used for hybridizing Affymetrix chips (GeneChip® Human Transcriptome Array 2.0(HTA2.0)). Data were normalised by gcRMA method and raw p-values adjusted by Bonferroni procedure (1%).
Project description:Whole exome sequencing of 5 HCLc tumor-germline pairs. Genomic DNA from HCLc tumor cells and T-cells for germline was used. Whole exome enrichment was performed with either Agilent SureSelect (50Mb, samples S3G/T, S5G/T, S9G/T) or Roche Nimblegen (44.1Mb, samples S4G/T and S6G/T). The resulting exome libraries were sequenced on the Illumina HiSeq platform with paired-end 100bp reads to an average depth of 120-134x. Bam files were generated using NovoalignMPI (v3.0) to align the raw fastq files to the reference genome sequence (hg19) and picard tools (v1.34) to flag duplicate reads (optical or pcr), unmapped reads, reads mapping to more than one location, and reads failing vendor QC.
Project description:Purpose: S100A10 uesed to located in the cell membrane or cytoplasm. However nuclear-located S100A10 was observed in the polyploid tumor giant cells(PGCCs) with daughter cells induced by cobalt chloride(CoCl2). To study the function of nuclear-located S100A10, we performed ChIP-Seq. Methods:LoVo and Hct116 cells were incubated in T25 flasks in RPMI-1640 medium until they reached 80-90% confluence. The cells were treated with 450 μM CoCl2 (Sigma-Aldrich, St. Louis, MO, USA) for 48-72h based on their resistance to hypoxia. After the treatment with CoCl2 was repeated for 3-4 times, there were 20-30% of PGCCs and 70-80% of small-sized daughter cells that originated from PGCCs among the total cells.ChIP assays were performed according to the instructions of the Pierce Magnetic ChIP Kit (Thermo Fisher Scientific, #26157) For data analysis of ChIP DNA, sequencing and library preparation were performed using Novogene Technology Co., Ltd. (Tianjin, China). Raw data (raw reads) of FASTQ format were first processed through in-house PERL scripts, and then reference genome and gene model annotation files were downloaded directly from the genome website. For a specific ChIP-seq binding site, individual reads were mapped to the plus or minus strand and presented significant enrichment. After mapping the reads to the reference genome, the MACS2 version 2.1.0 (model-based analysis of ChIP-seq) peak finding algorithm was used to identify regions of specific IP enrichment over background. A p-value threshold of enrichment of 0.05 was used for all data sets. The interaction between transcription factor or chromatin histone modification and DNA was not random, but they showed some specific sequence preference. MEME 52 and DREME 53 were used to detect the sequence motif, which was used to detect long and short consensus sequences. The position of peak summit around the transcript start sites of genes can predict the interaction sites of proteins and genes. Genes associated with different peaks were identified, and GO and KEGG enrichment analyses were performed. Results:The results showed that 4148 significant ChIP-seq peaked in Hct116-derived PGCCs with daughter cells, and 1380 peaked in LoVo-derived PGCCs with daughter cells. Genes with overlapping peaks were then enriched by Gene Ontology [GO], which revealed that these genes participated in various processes including stimulus, cell migration, and cell motility.Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed the potential targets involved in metabolic pathways, axon guidance TGF-β signaling pathway, and so on. Conclusions: S100A10 can be modified by SUMOylation in PGCCs and their daughter cells induced by either CoCl2 or chemotherapeutic drugs. SUMO1-modified S100A10 can translocate to the nucleus and regulate the expression of ARHGEF18, PTPRN2, and DEFA3 to promote the proliferation, migration, and invasion of PGCCs and their daughter cells, which may contribute to the poor prognosis in patients with cancer. The discovery of detailed mechanisms of the S100A10 SUMOylation, such as the specific lysine sites involved in SUMOylation and which SUMO E3 ligase regulates the SUMOylation of S100A10 requires further study.
Project description:The application of gene expression profiling is to investigate the pathogenesis of aggressive NFPAs. And to identify biomarkers that could play a key role in aggressive behaviour of NFPAs Gene expression was measured among pituitary glands, non-invasion NFPAs and invasion NFPAs
Project description:We use nucleosome maps obtained by high-throughput sequencing to study sequence specificity of intrinsic histone-DNA interactions. In contrast with previous approaches, we employ an analogy between a classical one-dimensional fluid of finite-size particles in an arbitrary external potential and arrays of DNA-bound histone octamers. We derive an analytical solution to infer free energies of nucleosome formation directly from nucleosome occupancies measured in high-throughput experiments. The sequence-specific part of free energies is then captured by fitting them to a sum of energies assigned to individual nucleotide motifs. We have developed hierarchical models of increasing complexity and spatial resolution, establishing that nucleosome occupancies can be explained by systematic differences in mono- and dinucleotide content between nucleosomal and linker DNA sequences, with periodic dinucleotide distributions and longer sequence motifs playing a secondary role. Furthermore, similar sequence signatures are exhibited by control experiments in which genomic DNA is either sonicated or digested with micrococcal nuclease in the absence of nucleosomes, making it possible that current predictions based on highthroughput nucleosome positioning maps are biased by experimental artifacts. Included are raw (eland) and mapped (wig) reads. The mapped reads are provided in eland and wiggle formats, and the raw reads are included in the eland file. This series includes only Mnase control data. The sonicated control is part of this already published accession, as is a in vitro nucleosome map: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE15188 We also studied data (in vitro and in vivo maps as well as a model) from http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE13622 and from: http://www.ncbi.nlm.nih.gov/sra/?term=SRA001023