Analysis on switching of alternative exons 1 causing aberrant expression of estrogen synthetase

我们已经证明，乳腺癌组织近端区域芳香酶表达的升高与这些区域人类芳香酶基因的组织特异性外显子1的切换显著相关。为进一步探讨乳腺癌组织中芳香酶基因转录因子的外显子1b向外显子1c转换的原因，以培养的乳腺脂肪基质细胞为材料，观察了不同因素对芳香酶基因外显子1表达水平和选择性的影响。在含小牛血清培养的基质细胞中，芳香酶基因从外显子1b转录而来。然而，剥夺血清会导致芳香酶mRNA的迅速升高，并导致芳香酶转录产物切换到外显子1c。此外，还从外显子1b转录了HepG2细胞和非恶性乳腺组织中的芳香酶mRNA。然后，使用包含4个主要可选外显子1和启动子的新报告，用荧光启动子分析检测了负责外显子1b在HepG2细胞中特异性利用的启动子区。结果表明，决定外显子1b在细胞内优先利用的转录元件位于外显子1b的启动子区-255~-1145。我们还观察到在骨和血管组织中有显著水平的免疫反应芳香酶蛋白和芳香酶mRNA。由于这些组织中雌激素水平的降低与骨组织中的骨质疏松和血管组织中的动脉硬化有关，而且这些组织中的芳香化酶mRNA是从外显子1b转录而来的，芳香酶基因的替代外显子1的转换可能是导致这些疾病的部分原因。

We have shown that elevation of aromatase expression in the proximal regions to breast cancer tissues was significantly correlated with switching of tissue-specific exons 1 of human aromatase gene in these regions. To further investigate what causes elevation of the aromatase mRNA and switching from exon 1b to exon 1c in the transcription factors of the aromatase gene in human breast cancer tissues, the effects of various on the exoression levels and preference of alternative expns 1 were examined in cultured adipose stromal cells from breast tissues. The aromatase mRNA was transcribed from exon 1b in the stromal cells cultured in the presence of calf serum. However, deprivation of the serum induced rapid elevation of aromatase mRNA and switching of aromatase transcripts to exon 1c the cells. Aromatase mRNA in HepG2 cells as well as in non-malignant breast tissues was also transcribed from exon 1b.Then, the promoter region responsible for the exon 1b-specific utilization in HepG2 cells was examined by fluorometric promoter assay using a new reporter containing 4 major alternative exons 1 and promoters. The results suggested that transcriptional elements determining preferential utilization of exon 1b in the cells was located on the promoter region of exon 1b form -255 to -1145. We have also observed significant levels of immunoreactive aromatase proteins and aromatase mRNA in the bone and vascular tissues. As decreased levels of estrogen in these tissues are paid attention in connecion with causes of osteoporosis in bone tissues and arterial sclerosis in vascular tissues and aromatase mRNA in these tissues is transcribed from exon 1b, switching of alternative exons 1 of the aromatase gene might contribute to a part of causes in these diseases.