Hayes Nandini V L, Blackburn Edith, Smart Laura V, Boyle Mary M, Russell Graham A, Frost Teresa M, Morgan Byron J T, Baines Anthony J, Gullick William J
Cancer Biology Laboratory, Research School of Biosciences, Centre for Biomedical Informatics, Institute of Mathematics and Statistics, University of Kent, Canterbury, UK.
Clin Cancer Res. 2007 Jun 1;13(11):3147-55. doi: 10.1158/1078-0432.CCR-06-2237.
The neuregulin (NRG) 1, 2, and 3 genes undergo extensive alternative mRNA splicing, which results in variants that show structural and functional diversity. The aims of this study were to establish whether the fourth member of this family, NRG4, is expressed in prostate cancer, if it is alternatively spliced and whether any functional differences between the variants could be observed.
The expression of NRG4 was determined using immunohistochemical staining of 40 cases of primary prostate cancer. Bioinformatic analysis and reverse transcription-PCR (RT-PCR) using NRG4 isotype-specific primers on a panel of normal and prostate cancer cell lines were used to identify alternatively spliced NRG4 variants. Expression of these variants was determined using isotype-specific antibodies. Transfection into Cos-7 cells of two of these green fluorescent protein-tagged variants allowed analysis of their subcellular location. Four of the variants were chemically synthesized and tested for their ability to activate the ErbB4 receptor.
NRG4 was variably expressed in the cytoplasm in the majority of prostate cancer cases, and in a subset of cases in the membrane, high levels were associated with advanced disease stage. Four novel NRG4 splice variants (NRGA2, NRG4 B1-3) were characterized, where each seemed to have a different subcellular location and were also expressed in the cytoplasm of the prostate tumors. NRG4 B3 was also present in endothelial cells. In transfected cells, the A type variant (NRG4 A1) was localized to the membrane, whereas the B type variant (NRG4 B1), which lacks the predicted transmembrane region, had an intracellular localization. Only the variants with an intact epidermal growth factor-like domain activated ErbB4 signaling.
NRG4 overexpression is associated with advanced-stage prostate cancer. The alternative splice variants may have different roles in cell signaling, some acting as classic receptor ligands and some with as-yet unknown functions.
神经调节蛋白（NRG）1、2和3基因经历广泛的可变mRNA剪接，产生具有结构和功能多样性的变体。本研究的目的是确定该家族的第四个成员NRG4是否在前列腺癌中表达，是否发生可变剪接，以及是否能观察到变体之间的任何功能差异。
使用40例原发性前列腺癌的免疫组织化学染色来确定NRG4的表达。利用生物信息学分析以及在一组正常和前列腺癌细胞系上使用NRG4同种型特异性引物进行逆转录聚合酶链反应（RT-PCR），以鉴定可变剪接的NRG4变体。使用同种型特异性抗体确定这些变体的表达。将其中两个带有绿色荧光蛋白标签的变体转染到Cos-7细胞中，以分析它们的亚细胞定位。化学合成了其中四个变体，并测试它们激活ErbB4受体的能力。
在大多数前列腺癌病例中，NRG4在细胞质中呈可变表达，在一部分病例中在细胞膜上表达，高水平与疾病晚期相关。鉴定出四种新的NRG4剪接变体（NRGA2、NRG4 B1 - 3），每种变体似乎具有不同的亚细胞定位，并且也在前列腺肿瘤的细胞质中表达。NRG4 B3也存在于内皮细胞中。在转染细胞中，A型变体（NRG4 A1）定位于细胞膜，而缺乏预测跨膜区域的B型变体（NRG4 B1）具有细胞内定位。只有具有完整表皮生长因子样结构域的变体激活ErbB4信号传导。
NRG4过表达与晚期前列腺癌相关。可变剪接变体可能在细胞信号传导中具有不同作用，一些充当经典受体配体，一些具有尚未明确的功能。
