Epigenomic remodeling of the PAX8 cistrome in high grade serous ovarian cancer [RNA-Seq]
Ontology highlight
ABSTRACT: We report mapping of the PAX8 cistrome in three high grade serous ovarian cancer cell lines (KURAMOCHI, OVSAHO, and JHOS4) compared to three benign immortalized fallopian tube cell lines (FT33, FT194, and FT246). We identified a highly conserved PAX8 binding pattern common across benign fallopian tube cell lines that was distinct from the unique PAX8 binding patterns seen in each cancer cell line. Comparison of benign and malignant Mullerian cell lines with and without PAX8 knockdown. For each cell line, three distinct siRNAs targeting PAX8 plus a pool of all three siRNAs were examined and compared to both a non-transfected control as well as a control transfected with a non-targeting siRNA.
Project description:We report mapping of the PAX8 cistrome in three high grade serous ovarian cancer cell lines (KURAMOCHI, OVSAHO, and JHOS4) compared to three benign immortalized fallopian tube cell lines (FT33, FT194, and FT246). We identified a highly conserved PAX8 binding pattern common across benign fallopian tube cell lines that was distinct from the unique PAX8 binding patterns seen in each cancer cell line. Comparison of benign and malignant Mullerian cell lines.
Project description:We report mapping of the PAX8 cistrome in three high grade serous ovarian cancer cell lines (KURAMOCHI, OVSAHO, and JHOS4) compared to three benign immortalized fallopian tube cell lines (FT33, FT194, and FT246). We identified a highly conserved PAX8 binding pattern common across benign fallopian tube cell lines that was distinct from the unique PAX8 binding patterns seen in each cancer cell line.
Project description:We report mapping of the PAX8 cistrome in three high grade serous ovarian cancer cell lines (KURAMOCHI, OVSAHO, and JHOS4) compared to three benign immortalized fallopian tube cell lines (FT33, FT194, and FT246). We identified a highly conserved PAX8 binding pattern common across benign fallopian tube cell lines that was distinct from the unique PAX8 binding patterns seen in each cancer cell line.
Project description:The goal of this experiment was to identify genes that were expressed at higher levels in benign human mammary epithelial cells than in breast cancer cell lines and that were induced by 5AZA treatment in breast cancer cell lines. Six breast cancer cell lines were selected for demethylation studies based on known tumor suppressor gene expression regulation by promoter region hypermethylation: HCC1569 (CCND2), HCC1954 (SCGB3A1, APC, RASSF1A), MCF-7 (RAR-beta2), MDA-MB-231 (ESR1), UACC3199 (BRCA1), and BT-549 (hypermethylator phenotype). Other than MCF10A we specifically avoided immortalized benign human mammary epithelial cell lines for this experiment as these cells frequently show tumor suppressor gene methylation (e.g. p16) and gene expression profiles that are intermediate between normal breast epithelial cells and breast cancer. Instead, we opted to test six first-passage benign human mammary epithelial cell cultures (HME) generated in serum-free media from small fragments of normal breast tissue obtained from young women undergoing fibroadenoma excision. The 5AZA dose (0.5 microM) was selected based on evaluation of growth curves and induction of BNC1, SERPINB, and TKTL1 gene expression measured by RT-PCR in benign and malignant cells. The breast cancer cell lines, HME cultures, and MC10A cells were treated with 0.5 microM 5AZA (Sigma-Aldrich, St. Louis, MO) in DMSO or DMSO alone for six days after which the cells were harvested, and RNA prepared using the Illumina TotalPrep kit (AMIL1791, Life Technologies, Grand Island, NY). Whole genome expression was assessed using the Illumina HumanWG-6-v3 chip Gene expression was evaluated in 6 breast cancer cell lines, 6 primary breast epithelial cell cultures, and MCF10A cells after 6 days in DMSO or DMSO plus 0.5 microM 5AZA.
Project description:To identify binding partners of Salmonella’s thioredoxin protein encoded by the trxA gene, we performed tandem-affinity purification (TAP) using a C-terminal fusion of thioredoxin with calmodulin-binding peptide and Protein A. TrxA-binding molecules were identified by mass spectrometry (MS) analysis
Project description:Objective of this work was to characterize the miRNA profile of exosomes isolated from brain-homing cell lines (MDA-MB-231BR, CTC1BMSM, and 70W) with their respective parental non-brain metastatic cell lines (MDA-MB-231P, CTC1P and MeWo). Exosomes derived from the six cell lines were isolated. Brain metastatic cell-derived exosomal miRNAs were compared with non brain metastatic cell lines (MDA-MB-231BR versus MDA-MB-231P, CTC1BMSM versus CTC1P, and 70W versus MeWo).
Project description:RRP1B is a Metastasis Modifier that Regulates the Transcriptome through mRNA Splicing To assess the role of RRP1B in alternative mRNA splicing, we knocked down the expression of Rrp1b in the highly metastatic mouse mammary tumor cell line Mvt-1 using shRNA. Two control and two knockdown stable cell lines were selected for RNA-sequencing.
Project description:The goals of this study were to identify LIN28 downstream gene targets in breast cancer cells. We use a subclone of the MCF-7 breast cancer cell line, MCF-7M as our model system. Methods: mRNA profiles from MCF-7M breast cancer cells treated with siRNA against non-targeting control (NT), LIN28, hnRNP A1, LIN28 and hnRNPA1 (LIN28A1) for 72 hrs were generated by deep sequencing, in duplicate, using Illumina HiSeq 2000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays Results: Using an optimized data analysis workflow, we mapped over 200 million sequence reads per sample to the human genome (build h19). Each of the four groups had two biological replicates. We developed a custom method to identify alternative splicing events and identified 111 genes with significant (FDR<0.05) differential splicing for LIN28 depleted cells compared to non-targeting siRNA control, as well as 249 and 182 genes for hnRNP A1 and LIN28A1 respectively. RNA-seq data were validated with by qRT–PCR analysis of a subset of genes. Conclusions: Results reveal that LIN28 regulates alternative splicing and steady state mRNA expression of genes implicated in aspects of breast cancer biology. Notably, cells lacking LIN28 undergo significant isoform switching of the ENAH gene, resulting in a decrease in the expression of ENAH exon 11a isoform. Expression of ENAH isoform 11a has been shown to be elevated in breast cancers that express HER2. mRNA profiles of MCF-7M cells treated with siRNA for NT control, LIN28, hnRNP A1, and LIN28 plus hnRNP A1 (A1) (LIN28A1) were generated by deep sequencing, in duplicate, using Illumina HiSeq 2000
Project description:To investigate the role for LSD1 under hypoxia condition. we depleted LSD1 gene with siRNA in Huh-1 cell lines under 1% O2 hypoxia condtion, and than perforemed gene expression microarray analysis. Using Gene Set Enrichment Analysis (GSEA), determined to identify the biological pathway. Determined the gene expression profile of the LSD konckdown effect under hypoxia condition. Using Gene Set Enrichment Analysis (GSEA) decided to identify the biological pathways.