Unknown,Transcriptomics,Genomics,Proteomics

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Effect of MEL-18 knockdown on estrogen-sensitive MCF7 breast cancer cells

ABSTRACT: Gene expression analysis of MEL-18-silenced MCF7 cell lines. MEL-18 is a component of the polycomb repressive complex (PRC)-1, which is a critical epigenetic modulator of stem cell regulation and normal and cancerous development. Accumulating studies have suggested that MEL-18 might act as a tumor suppressor in several human tumors, including breast cancer. Results provide insight into the functional role of MEL-18 in estrogen-dependent breast cancer. MCF7 cells stably infected with lentiviruses encoding either control (shCon) or MEL-18 shRNA (shMEL) were cultured in phenol-red free DMEM supplemented with 10% FBS for 48 h. Total RNA was isolated from the cultures using Trizol reagent. For each of the 2 conditions, 2 biological replicates were included. In total, 4 microarray samples were analyzed; 2 controls and 2 shRNA MEL-18 knockdowns. All labeling, hybridization and scanning steps were performed according to the manufacturers’ instructions.

ORGANISM(S): Homo sapiens

SUBMITTER: Gu Kong

PROVIDER: E-GEOD-64716 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Bmi-1 and Mel-18 are close structural homologues that belong to the polycomb group (PcG) of transcriptional regulators of homeotic gene expression in development. They are believed to stably maintain repression of gene expression by altering the state of chromatin at specific promoters. A number of clinical and experimental observations have also implicated Bmi-1 in tumorigenesis and stem cell maintenance. Bmi-1 overexpression or amplification has been observed in a number of human malignancies, particularly in B-cell lymphomas, medulloblastomas and breast cancer. We report here that shRNA-mediated knock-down of either Bmi-1 or Mel-18 in human medulloblastoma DAOY cells results in the inhibition of proliferation, loss of clonogenic survival and anchorage-independent growth, and suppression of xenograft tumor formation in nude mice. Furthermore, overexpression of both Bmi-1 and Mel-18 significantly increased clonogenic survival of Rat1 fibroblasts. In contrast, stable downregulation of Bmi-1 or Mel-18 alone did not affect the growth of SK-OV-3 or U2OS cancer cell lines or normal human WI38 fibroblasts. Gene expression analysis of shRNA-expressing DAOY cells has demonstrated a significant overlap in the Bmi-1- and Mel-18-regulated genes and revealed novel gene targets under their control. Taken together, these results suggest that Bmi-1 and Mel-18 might have overlapping functions in human tumorigenesis. Experiment Overall Design: DAOY cells were transduced with the indicated lentiviral shRNA and stable cell lines were generated by selection in puromycin (1 Âµg/ml). 2 x 105 cells of each stable cell line were plated onto 60 mm dishes in triplicates and harvested 4 days later. Triplicate total RNA samples from shRNA-expressing and mock-transduced DAOY cells were isolated using affinity resin (Qiagen RNeasy Mini Kit, Qiagen AG). RNA Integrity and purity were assessed with the RNA 6000 Nano LabChip system on Bioanalyzer 2100 (Agilent Technologies). Gene array analysis was conducted at the Genomics Factory at Novartis PHARMA AG, Basel, Switzerland using Gene Chip Human Genome 133 2.0 Plus Expression Array (Affymetrix Inc.).

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Effect of MEL-18 knockdown on estrogen-sensitive MCF7 breast cancer cells

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