Systems Biology Ireland, School of Medicine, University College Dublin, Belfield, D04 V1W8 Dublin 4, Ireland.
Drug Research Program, Faculty of Pharmacy, University of Helsinki, 00014 Helsinki, Finland.
Biomolecules. 2022 Jul 17;12(7):993. doi: 10.3390/biom12070993.
Alternative mRNA splicing is common in cancers. In BRAF V600E-mutated malignant melanoma, a frequent mechanism of acquired resistance to BRAF inhibitors involves alternative splicing (AS) of BRAF. The resulting shortened BRAF protein constitutively dimerizes and conveys drug resistance. Here, we have analysed AS in SK-MEL-239 melanoma cells and a BRAF inhibitor (vemurafenib)-resistant derivative that expresses an AS, shortened BRAF V600E transcript. Transcriptome analysis showed differential expression of spliceosome components between the two cell lines. As there is no consensus approach to analysing AS events, we used and compared four common AS softwares based on different principles, DEXSeq, rMATS, ASpli, and LeafCutter. Two of them correctly identified the BRAF V600E AS in the vemurafenib-resistant cells. Only 12 AS events were identified by all four softwares. Testing the AS predictions experimentally showed that these overlapping predictions are highly accurate. Interestingly, they identified AS caused alterations in the expression of melanin synthesis and cell migration genes in the vemurafenib-resistant cells. This analysis shows that combining different AS analysis approaches produces reliable results and meaningful, biologically testable hypotheses.
可变剪接在癌症中很常见。在 BRAF V600E 突变型恶性黑色素瘤中，获得性 BRAF 抑制剂耐药的常见机制涉及 BRAF 的可变剪接 (AS)。由此产生的缩短的 BRAF 蛋白持续二聚化并传递耐药性。在这里，我们分析了 SK-MEL-239 黑色素瘤细胞和表达 AS、缩短的 BRAF V600E 转录本的 BRAF 抑制剂 (vemurafenib) 耐药衍生物中的 AS。转录组分析显示两种细胞系之间剪接体成分的差异表达。由于没有分析 AS 事件的共识方法，我们使用并比较了基于不同原理的四种常见 AS 软件，即 DEXSeq、rMATS、ASpli 和 LeafCutter。其中两种软件正确识别了 vemurafenib 耐药细胞中的 BRAF V600E AS。只有四种软件中的 12 个 AS 事件被识别。通过实验测试 AS 预测表明，这些重叠的预测具有高度准确性。有趣的是，它们鉴定出 AS 导致 vemurafenib 耐药细胞中黑色素合成和细胞迁移基因表达的改变。这项分析表明，结合不同的 AS 分析方法可产生可靠的结果和具有生物学意义、可测试的假说。
