Increasing the relative expression of endogenous non-coding Steroid Receptor RNA Activator (SRA) in human breast cancer cells using modified oligonucleotides

Products of the Steroid Receptor RNA Activator gene (SRA1) have the unusual property to modulate the activity of steroid receptors and other transcription factors both at the RNA (SRA) and the protein (SRAP) level. Balance between these two genetically linked entities is controlled by alternative splicing of intron-1, whose retention alters SRAP reading frame. We have previously found that both fully-spliced SRAP-coding and intron-1-containing non-coding SRA RNAs co-exist in breast cancer cell lines. Herein, we report a significant (Student's t-test, P < 0.003) higher SRA–intron-1 relative expression in breast tumors with higher progesterone receptor contents. Using an antisense oligoribonucleotide, we have successfully reprogrammed endogenous SRA splicing and increased SRA RNA–intron-1 relative level in T5 breast cancer cells. This increase is paralleled by significant changes in the expression of genes such as plasminogen urokinase activator and estrogen receptor beta. Estrogen regulation of other genes, including the anti-metastatic NME1 gene, is also altered. Overall, our results suggest that the balance coding/non-coding SRA transcripts not only characterizes particular tumor phenotypes but might also, through regulating the expression of specific genes, be involved in breast tumorigenesis and tumor progression

The steroid receptor RNA activator protein is expressed in breast tumor tissues

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Identification of an octamer-binding site controlling the activity of the small breast epithelial mucin gene promoter

International audienceTABLE OF CONTENTS 1. Abstract 2. Introduction 2. Material and Methods 2.1. Cell Culture 2.2. RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) 2.3. Rapid amplification of the 5'-cDNA end (RACE) 2.4. Identification of transcription factors binding sites within the 87-bp enhancer region (ENH) 2.5. Plasmids 2.6. Transient DNA transfections and luciferase assays 3. Results 3.1. Identification of the transcription initiation sites 3.2. Endogenous SBEM promoter activity in mammary and non-mammary cancer cells 3.3. Analysis of SBEM promoter activity in mammary and non-mammary cancer cells 3.4. The ENH region (-357/-270) drives a strong breast-specific promoter activity 3.5. Importance of octamer-binding transcription factors motif in the SBEM promoter activity 3.6. Oct1 and Oct2 enhance both exogenous and endogenous SBEM promoter activities 4. Discussion 5. Acknowledgement 6. References 1. ABSTRACT The human small breast epithelial mucin (SBEM) gene has been identified as being preferentially expressed in mammary epithelial cells and over-expressed in breast tumors. In this report, we have characterized the promoter of SBEM gene in order to identify sequences responsible for this strong mammary expression. A series of SBEM promoter/luciferase constructs were transiently transfected into both breast (MCF-7, BT-20) and non-breast (HeLa and HepG2) cell lines. In addition to the minimal promoter and to a repressor region, we have identified an 87-bp sequence (-357/-270) driving a strong breast-specific expression. Site-directed mutagenesis of a putative octamer-binding transcription factor binding site located within this latter region led to a strong decrease of the transcriptional activity of the SBEM promoter. Furthermore, transient over-expression of Oct1 and Oct2 not only increased SBEM promoter reporter activity, but also enhanced endogenous SBEM mRNA level. Overall, the data suggest that octamer-binding transcription factors participate in the strong expression of SBEM gene in breast tissues. Clarifying the SBEM gene regulation will help to dissect mechanisms underlying transcription of normal breast and breast cancer-associated genes

Alternative Splicing of the First Intron of the Steroid Receptor RNA Activator (SRA) Participates in the Generation of Coding and Noncoding RNA Isoforms in Breast Cancer Cell Lines

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