Artikel
Identification of novel estrogen receptor beta splice variants in MDA-MB-231 breast cancer cells
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Veröffentlicht: | 20. März 2006 |
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Gliederung
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Objective: After identification of human estrogen receptor (ER) β gene and its first transcript, several splice variants of this mRNA have been identified. Given that function of many ERβ isoforms remains unclear, we intended to clone full-length ERβ isoforms for subsequent function analyzes by means of heterologous gene expression studies.
Methods: Reversely transcribed total RNA from MDA-MB-231 cells was amplified using the PCR primer pair 5´-CAAGGCCGGTGTGTTTATCT-3´ and 5´-GGCGTCACTGAGACTGTGG-3´. After standard agarose gel electrophoresis, cDNA amplicons were eluted, sequenced, and inserted into pTARGET expression vector. COS-1 cells were transfected using Transfectin reagent (BioRad), stable clones were generated by G418 selection. Relative cell numbers were measured by means of the Cell titer blue Assay (Promega), migration was analyzed by standard wound healing assays, and cellular apoptosis was examined both by measurement of caspase 3/7 activation and by annexin V / propidium iodide staining.
Results: We isolated two novel ERβ exon deletion variants, delta 1/2/5, and delta 1/2/5/6 lacking the ERβ1 exons number 1, 2 and 5 or 1, 2, 5 and 6 respectively. Specific RT-PCR primer pairs for detection of these splice variants binding at the border of exon 0 and 3 and the border of exon 3 and 6 or exon 3 and 7 respectively enabled us to verify the expression of both novel splice variants in other tissues and tumor cell lines. COS-1 cells stably transfected with ERβ1 or the novel ERβ isoforms exhibited a slowed proliferation and migration, but a significantly increased basal apoptosis rate even in the absence of ligand estradiol.
Conclusion: Like ERβ1, the novel exon skipped ERβ1 splice variants are able to affect COS-1 cell growth, apoptosis and migration in a ligand-independent manner. Whether our work is able to support previous studies suggesting ERβ isoforms acting as tumor suppressor is currently investigated in further heterologous gene expression studies in human tumor cell lines.