MicroRNA expression data from leukemia and breast cancer cell lines with/without treatment with microparticles
ABSTRACT: Microparticles (MPs) comprise the major source of systemic RNA including microRNA (miRNA), the aberrant expression of which appears to be associated with stage, progression and spread of many cancers. We have shown MPs to transfer multidrug resistance proteins accross both haematological and and non-haematological cancers. using microarray miRNA profiling analysis we now analyze changes in miRNA profiles of both cancer types following microparticle transfer. We identified certain upregulated miRNAs in both cancer types. Total RNA was extracted and pooled from duplicate experiments for hybridization on Affymetrix microarrays from (i) the parental drug sensitive leukaemia (CEM) or breast cancer (MCF-7) cells, (ii) their Multidrug Resistant strains leukaemia (VLB100) or breast cancer ( DX cells), (iii) the microparticles isolated from the resistant cells: VLBMP or DXMP, and (iv) the cocultured samples: sensitive cell co-incubated with MPs from their resistant cells ( leukaemia: CEM+VLBMP) or(breast cancer: MCF-7+DXMP). We sought to examine the miRNA profiles of the drug sensitve cells after MP transfer from drug resistant cells across leukaemia nd breact cancer cell lines.
Project description:Cancer patients often have an activated clotting system and are at increased risk for venous thrombosis. In this study, we analyzed tissue factor (TF) expression in four different human pancreatic tumor cell lines for the purpose of producing derivative tumors in vivo. We found that two of the lines expressed TF and released TF-positive microparticles (MPs) into the culture medium. The majority of TF protein in the culture medium was associated with MPs. Importantly, only TF-positive cell lines activated coagulation in nude mice, and this activation was abolished by an anti-human TF antibody. Of the two TF-positive lines only one produced detectable levels of human MP TF activity in the plasma when grown orthotopically in nude mice. Surprisingly, <5% of human TF protein in plasma from tumor-bearing mice was associated with MPs. Mice with TF-positive tumors and elevated levels of circulating TF-positive MPs had increased thrombosis in a saphenous vein model. In contrast, we observed no difference in thrombus weight between tumor bearing and control mice in an inferior vena cava stenosis model. Our studies suggest that in a xenograft mouse model tumor TF activates coagulation, whereas TF on circulating MPs may trigger venous thrombosis. Overall design: 8 human tumor pancreatic cell lines
Project description:To identify the miRNA expressing profiles of Platelet microparticles（PMPs, we have employed the Agilent Human miRNA 8×60K (Design ID:046064) microarray. Platelet microparticles. The platelets were derived from citrated blood of healthy human donors under an Institutional Review Board-approved protocol. Platelets were isolated after centrifugation of blood (1200r for 30 min at 21℃), then the supernatant (platelet-rich plasma) was centrifuged at 2000r for 30 min at 21℃, and the pellet containing platelets was resuspended in RPMI-1640 medium (HyClone, Logan, UT). Platelets were counted (Clinical Laboratory, Shanghai First Maternity and Infant Hospital, Shanghai) and adjusted to a density of 150 × 106 cells/mL before supplement with 1.5% ACD(sigma ) and stimulated with thrombin (1.0 u/mL; Takeda Austria) for 1 h. PMPs were in the supernatant after centrifugation at 4000r for 10 min at 4℃,then the supernatants were centrifuged at 50,000 × g for 60 min at 4 °C. The pellets containing MPs were resuspended in RPMI-1640 medium and quantified by BCA method. The gene expressions of three independent paired PMPs from platelets stimulated by thrombin or apoptosis.
Project description:Expression of estrogen receptor (ESR1) determines whether a breast cancer patient receives endocrine therapy as part of their adjuvant care, but does not guarantee patient response. However, the molecular factors that define endocrine response in ESR1-positive breast cancer patients remain poorly understood. Here, we characterize the DNA methylome of endocrine sensitivity and demonstrate the potential impact of differential DNA methylation on endocrine response in breast cancer. We show that DNA hypermethylation occurs predominantly at estrogen-responsive enhancers and is associated with reduced ESR1 binding and decreased gene expression of key regulators of ESR1-activity; thus providing a novel mechanism by which endocrine response is abated in ESR1-positive breast cancers. Conversely, we delineate that ESR1-responsive enhancer hypomethylation is critical in transition from normal mammary epithelial cells to endocrine responsive ESR1-positive cancer. Cumulatively these novel insights highlight the potential of ESR1-responsive enhancer methylation to both predict ESR1-positive disease and stratify ESR1-positive breast cancer patients as responders to endocrine therapy. Methylation profiling with Illumina's HumanMethylation450K array was performed on ESR1-positive hormone sensitive MCF7 cells, and three different well characterised endocrine resistant MCF7-derived cell lines; tamoxifen-resistant (TAMR), fulvestrant-resistant (FASR) and estrogen deprivation resistant (MCF7X) cells. For each cell line two biological replicates were profiled bringing the number of samples to eight.
Project description:The stem cell gene LIN28B was recently shown to be overexpressed in a foetal-like subgroup of juvenile myelomonocytic leukaemia. Given the involvement of LIN28B in a variety of solid paediatric cancers, we conducted a meta-analysis of LIN28B levels using publicly available gene expression data of 1361 paediatric leukaemia samples. Interestingly, this analysis revealed LIN28B overexpression in 102 childhood leukaemia patients (7.5%), suggesting oncogenic activity for LIN28B in the context of paediatric haematological diseases. As the mode of action of LIN28B during normal and malignant haematopoiesis remains largely unexplored, we subsequently analysed the transcriptional consequences of LIN28B modulation on normal and malignant haematopoietic cells and identified the long non-coding RNA (lncRNA) H19 as the first LIN28B-regulated lncRNA. Oci-AML3 cells were retrovirally transduced with MSCV-PIG-LIN28B and MSCV-PIG-empty vectors (gifts from Johua Mendel lab), selected with puromycin and hybridized on Agilent microarray.
Project description:The stem cell gene LIN28B was recently shown to be overexpressed in a foetal-like subgroup of juvenile myelomonocytic leukaemia. Given the involvement of LIN28B in a variety of solid paediatric cancers, we conducted a meta-analysis of LIN28B levels using publicly available gene expression data of 1361 paediatric leukaemia samples. Interestingly, this analysis revealed LIN28B overexpression in 102 childhood leukaemia patients (7.5%), suggesting oncogenic activity for LIN28B in the context of paediatric haematological diseases. As the mode of action of LIN28B during normal and malignant haematopoiesis remains largely unexplored, we subsequently analysed the transcriptional consequences of LIN28B modulation on normal and malignant haematopoietic cells and identified the long non-coding RNA (lncRNA) H19 as the first LIN28B-regulated lncRNA. K562 cells were retrovirally transduced with shRNA GN36578 against LIN28B and a shRNA-miR non-targeting control TRH1103 (both Transomic), selected with puromycin and hybridized on Agilent microarray.
Project description:Retinoic acid (RA), the main active vitamin A metabolite, controls multiple biological processes such as cell proliferation and differentiation through genomic programs and kinase cascades activation. Due to these properties, RA has proven anti-cancer capacity. Several breast cancer cells respond to the antiproliferative effects of RA, while others are RA-resistant. However, the overall signaling and transcriptional pathways that are altered in such cells have not been elucidated. Here, in a large-scale analysis of the phosphoproteins and in a genome-wide analysis of the RA-regulated genes, we compared two human breast cancer cell lines, a RA-responsive one, the MCF7 cell line, and a RA-resistant one, the BT474 cell line, which depicts several alterations of the "kinome".
Project description:Background: Cancer stem cells are presumed to have virtually unlimited proliferative and self-renewal abilities and to be highly resistant to chemotherapy, a feature that is associated with overexpression of ATP-binding cassette transporters. We investigated whether prolonged continuous selection of cells for drug resistance enriches cultures for cancer stem-like cells. Methods: Cancer stem cells were defined as CD44+/CD24– cells that could self-renew (ie, generate cells with the tumorigenic CD44+/CD24– phenotype), differentiate, invade, and form tumors in vivo. We used doxorubicin-selected MCF-7/ADR cells, weakly tumorigenic parental MCF-7 cells, and MCF-7/MDR, an MCF-7 subline with forced expression of ABCB1 protein. Cells were examined for cell surface markers and side-population fractions by microarray and flow cytometry, with in vitro invasion assays, and for ability to form mammospheres. Xenograft tumors were generated in mice to examine tumorigenicity (n = 52). The mRNA expression of multidrug resistance genes was examined in putative cancer stem cells and pathway analysis of statistically significantly differentially expressed genes was performed. All statistical tests were two-sided. Results: Pathway analysis showed that MCF-7/ADR cells express mRNAs from ABCB1 and other genes also found in breast cancer stem cells (eg, CD44, TGFB1, and SNAI1). MCF-7/ADR cells were highly invasive, formed mammospheres, and were tumorigenic in mice. In contrast to parental MCF-7 cells, more than 30% of MCF-7/ADR cells had a CD44+/CD24– phenotype, could self-renew, and differentiate (ie, produce CD44+/CD24– and CD44+/CD24+ cells), and overexpressed various multidrug resistance-linked genes (including ABCB1, CCNE1, and MMP9). MCF-7/ADR cells were statistically significantly more invasive in Matrigel than parental MCF-7 cells (MCF-7 cells = 0.82 cell per field and MCF-7/ADR = 7.51 cells per field, difference = 6.69 cells per field, 95% confidence interval = 4.82 to 8.55 cells per field, P<.001). No enrichment in the CD44+/CD24– or CD133+ population was detected in MCF-7/MDR. Conclusion: The cell population with cancer stem cell characteristics increased after prolonged continuous selection for doxorubicin resistance. PARALLEL study design with 4 samples Parental MCF-7 cell line versus Doxorubicin Resistant MCF-7 cell sublines Biological replicates: 2 parental controls, 2 drug resistant, independently grown and harvested. agent:Selection agent is multi-step doxorubicin selection: MCF7226ng, MCF7262ng biological replicate: MCF71, MCF72 biological replicate: MCF226ng, MCF7262ng
Project description:Human breast cancer cell line MCF-7 is usually sensitive to chemotherapy drug BMS-554417, an insulin receptor (IR) and insulin-like growth factor receptor (IGFR) inhibitor. However, through step-wise increase in BMS-554417 doses in culture media, we were able able to screen and select a single MCF-7 clone that is BMS-554417 resistant. It is cross resistant to BMS-536924. This new line of MCF-7 cells was named as MCF-7R4. The transcriptome profiling of both MCF-7 and MCF-7R4 was performed using Affymetrix HG-U133 plus2.0 GeneChip arrays. Five replicates of MCF-7 and five replicates of MCF-7R4 were profiled.
Project description:Purpose: Development of resistance to tamoxifen is an important clinical issue in the treatment of patients with breast cancer. Tamoxifen resistance may be the result of the acquisition of epigenetic regulation such as DNA methylation within breast cancer cells resulting in changed mRNA expression of genes being pivotal for estrogen dependent growth. Alternatively, tamoxifen resistance may be due to selection of preexisting resistant cells, which may exhibit cancer stem-like characteristics or a combination of the two mechanisms. Methods: To evaluate the contribution of these possible mechanisms to tamoxifen resistance, we applied modified DNA methylation-specific digital karyotyping (MMSDK) and digital gene expression (DGE) in combination with massively parallel sequencing to analyze a well-established tamoxifen resistant cell line model: MCF-7/S0.5 (tamoxifen sensitive parental cell line) and 4 high-dosage tamoxifen selected resistant offspring sublines (MCF-7/TAMR-1, MCF-7/TAMR-4, MCF-7/TAMR-7 and MCF-7/TAMR-8). MMSDK uses BssHII as mapping enzyme (DNA methylation sensitive enzyme). Both MMSDK and DGE use NlaIII and MmeI to produce 20-21 bp tag. The indexed single-end sequencing was performed by Illumina HiSeq 2000 in BGI-Shenzhen. A dynamic programming algorithm-FASTX-Toolkit implemented in Perl was used to trim the adaptor sequence. The trimmed tags were subjected to quality filtering, so that only tags with sequencing quality higher than 30 for more than 80% of the nucleotides were used for subsequent analysis. For MMSDK tag mapping, we generated a simulated reference library, i.e., BssHII reference library, by in silico enzyme digestion of the human genome (hg19, UCSC) regardless of the methylation state. This library was used as reference for subsequent mapping of the tags in the MMSDK analysis. In the DGE analysis, refMrna (hg19, UCSC) was used as reference for mapping cDNA tags. Subsequently, the Burrows–Wheeler Aligner (BWA) procedure for aligning the MMSDK and DGE tags to the simulated BssHII reference library and refMrna reference library, respectively, was applied. Results: MMSDK libraries using BssHII/NlaIII were generated from the parental tamoxifen sensitive subline MCF-7/S0.5 and the 4 TAMR cell lines: TAMR-1, TAMR-4, TAMR-7 and TAMR-8. The 5 indexed MMSDK libraries were sequenced in one lane and 1.38 Gb clean tag data for all 5 cell lines were obtained, with an average sequencing amount of ~270 Mb per library. On average, 59.5 % of the tags with mapping quality ≥ 20 were mapped back to the simulated BssHII/NlaIII reference library. DGE libraries were also generated from MCF-7/S0.5 and the 4 TAMR cell lines. The 5 indexed DGE libraries were sequenced in one lane and obtained 1.71 Gb clean tag data for all 5 cell lines with an average sequencing amount of ~340 Mb per library. On average, 40.8 % with mapping quality ≥ 20 were mapped back to the simulated NlaIII human transcriptome (refMrna reference library). Our present study demonstrates large differences in global gene expression and DNA methylation profiles between parental tamoxifen-sensitive cell line and 4 high-dosage tamoxifen treatment selected resistant sublines. The tamoxifen resistant cell lines exhibited globally higher methylation level than the parental cell line and an inverse relationship between gene expression and DNA methylation in the promoter regions were noticed. High expression of SOX2 and alterations of other SOX gene family members, E2F gene family members and RB-related pocket protein genes as well as highlighted stem cell pathways imply that cancer initiating cells/stem cells are involved in the resistance to tamoxifen. DNA methylation and mRNA expression profiles from tamoxifen sensitive parental cell line MCF-7/S0.5 and 4 high dosage of tamoxifen selected resistant offspring sublines (MCF-7/TAMR-1, MCF-7/TAMR-4, MCF-7/TAMR-7 and MCF-7/TAMR-8) were analyzed by MMSDK and DGE methods, respectively, in combination of massively parallel sequencing, using Illumina HiSeq 2000
Project description:Tamoxifen is an effective anti-estrogen treatment for patients with estrogen receptor-positive (ER+) breast cancer. However, about 30% of such patients receiving tamoxifen as an adjuvant therapy experience recurrence within 15 years, and most patients with advanced disease eventually develop resistance to tamoxifen. To elucidate the underlying molecular mechanisms of tamoxifen resistance, we performed a systematic analysis of miRNA-mediated gene regulation in three clinically-relevant tamoxifen-resistant human breast cancer cell lines (TamRs) compared to their parental tamoxifen-sensitive MCF-7/S0.5 cell line. Alterations in the expression of 131 miRNAs in tamoxifen-resistant vs. parental cell lines were identified, 22 of which were common to all TamRs using both sequencing and LNA-based quantitative PCR technologies. ER+ and tamoxifen sensitive breast cancer cell line (MCF-7/S0.5) and its derived tamoxifen resistant clones: TAMR-1, TAMR-4 and TAMR-8 were miRNA expression profiled in triplicates of each using Exiqon's miRCURY LNA based microRNA Ready-to-use PCR, Human panel I+II, V2.R (Exiqon, product number 203608).