Wang Li, Gong Shuaishuai, Zhang Xi, Azhar Zeb, Chen Jialin
Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, Jiangsu, China.
Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, Jiangsu, China.
Gene. 2023 May 25;866:147330. doi: 10.1016/j.gene.2023.147330. Epub 2023 Mar 5.
The Androgen Receptor (AR) gene plays a key role in castration-resistant prostate cancer (CRPC). Controlling the progression of CRPC by inhibiting AR gene expression is one of the core directions for prostate cancer (Pca) drug development. A 23-amino acids retention, named exon 3a, into the DNA binding domain of the splice variant AR23 has been shown to prevent AR from entering the nucleus and restore the sensitivity of cancer cells to related therapies. In this study, we conducted a preliminary investigation of the splicing modulation of the AR gene in order to develop a splice-switching therapy for Pca by promoting exon 3a inclusion. Using mutagenesis-coupled RT-PCR with AR minigene and over-expression of certain splicing factors, we found that serine/arginine-rich (SR) proteins are key factors facilitating the recognition of the 3' splice site of exon 3a (L-3' SS), while the deletion or blocking of the polypyrimidine tract (PPT) region of the original 3' splice site of exon 3 (S-3' SS) could strongly enhance exon 3a splicing without affecting the function of any SR protein. Furthermore, we designed a series of antisense oligonucleotides (ASOs) to screen drug candidates, and ASOs targeting S-3' SS and its PPT region or the exonic region of exon 3 turned out to be most effective in rescuing exon 3a splicing. A dose-response test indicated ASO12 as the lead candidate drug significantly promoting the inclusion of exon 3a to more than 85%. MTT assay confirmed that the cell proliferation was significantly inhibited after ASO treatment. Our results provide the first glance to AR splicing regulation. With several promising therapeutic ASO candidates obtained here, further development of ASO drugs to treat CRPC is strongly encouraged.
雄激素受体（AR）基因在去势抵抗性前列腺癌（CRPC）中起关键作用。通过抑制AR基因表达来控制CRPC的进展是前列腺癌（Pca）药物研发的核心方向之一。已显示剪接变体AR23的DNA结合域中存在一个名为外显子3a的23个氨基酸的保留片段，可阻止AR进入细胞核并恢复癌细胞对相关疗法的敏感性。在本研究中，我们对AR基因的剪接调控进行了初步研究，旨在通过促进外显子3a的包含来开发一种针对Pca的剪接转换疗法。通过AR小基因的诱变偶联逆转录聚合酶链反应（RT-PCR）以及某些剪接因子的过表达，我们发现富含丝氨酸/精氨酸（SR）的蛋白质是促进外显子3a（L-3'SS）3'剪接位点识别的关键因素，而外显子3原始3'剪接位点（S-3'SS）的多嘧啶序列（PPT）区域的缺失或阻断可强烈增强外显子3a的剪接，且不影响任何SR蛋白的功能。此外，我们设计了一系列反义寡核苷酸（ASO）来筛选候选药物，结果表明靶向S-3'SS及其PPT区域或外显子3外显子区域的ASO在挽救外显子3a剪接方面最为有效剂量反应试验表明，作为主要候选药物的ASO12可将外显子3a的包含率显著提高至85%以上。MTT分析证实，ASO处理后细胞增殖受到显著抑制。我们的结果首次揭示了AR剪接调控情况。鉴于在此获得了几种有前景的治疗性ASO候选药物，强烈鼓励进一步开发用于治疗CRPC的ASO药物。
