Department of Biomedicine Neuroscience and Advanced Diagnostic, University of Palermo, Palermo, Italy.
McGill University Health Centre, Montréal, Canada.
J Exp Clin Cancer Res. 2024 Aug 5;43(1):217. doi: 10.1186/s13046-024-03139-z.
Aberrant alternative splicing events play a critical role in cancer biology, contributing to tumor invasion, metastasis, epithelial-mesenchymal transition, and drug resistance. Recent studies have shown that alternative splicing is a key feature for transcriptomic variations in colorectal cancer, which ranks third among malignant tumors worldwide in both incidence and mortality. Long non-coding RNAs can modulate this process by acting as trans-regulatory agents, recruiting splicing factors, or driving them to specific targeted genes. LncH19 is a lncRNA dis-regulated in several tumor types and, in colorectal cancer, it plays a critical role in tumor onset, progression, and metastasis. In this paper, we found, that in colorectal cancer cells, the long non-coding RNA H19 can bind immature RNAs and splicing factors as hnRNPM and RBFOX2. Through bioinformatic analysis, we identified 57 transcripts associated with lncH19 and containing binding sites for both splicing factors, hnRNPM, and RBFOX2. Among these transcripts, we identified the mRNA of the GTPase-RAC1, whose alternatively spliced isoform, RAC1B, has been ascribed several roles in the malignant transformation. We confirmed, in vitro, the binding of the splicing factors to both the transcripts RAC1 and lncH19. Loss and gain of expression experiments in two colorectal cancer cell lines (SW620 and HCT116) demonstrated that lncH19 is required for RAC1B expression and, through RAC1B, it induces c-Myc and Cyclin-D increase. In vivo, investigation from biopsies of colorectal cancer patients showed higher levels of all the explored genes (lncH19, RAC1B, c-Myc and Cyclin-D) concerning the healthy counterpart, thus supporting our in vitro model. In addition, we identified a positive correlation between lncH19 and RAC1B in colorectal cancer patients. Finally, we demonstrated that lncH19, as a shuttle, drives the splicing factors RBFOX2 and hnRNPM to RAC1 allowing exon retention and RAC1B expression. The data shown in this paper represent the first evidence of a new mechanism of action by which lncH19 carries out its functions as an oncogene by prompting colorectal cancer through the modulation of alternative splicing.
异常的剪接事件在癌症生物学中起着关键作用，导致肿瘤侵袭、转移、上皮-间充质转化和耐药性。最近的研究表明，剪接是结直肠癌转录组变异的一个关键特征，结直肠癌在全球范围内的发病率和死亡率均位居恶性肿瘤的第三位。长非编码 RNA 可以通过作为转录调节因子、招募剪接因子或驱动它们靶向特定基因来调节这个过程。LncH19 在多种肿瘤类型中失调，在结直肠癌中，它在肿瘤发生、进展和转移中起着关键作用。在本文中，我们发现，在结直肠癌细胞中，长非编码 RNA H19 可以与不成熟的 RNA 和剪接因子如 hnRNPM 和 RBFOX2 结合。通过生物信息学分析，我们鉴定了 57 个与 lncH19 相关的转录本，这些转录本含有与两个剪接因子 hnRNPM 和 RBFOX2 结合的位点。在这些转录本中，我们鉴定了 GTPase-RAC1 的 mRNA，其剪接异构体 RAC1B 在恶性转化中具有多种作用。我们在体外证实了剪接因子与转录本 RAC1 和 lncH19 的结合。在两种结直肠癌细胞系（SW620 和 HCT116）中进行的表达缺失和表达增益实验表明，lncH19 是 RAC1B 表达所必需的，并且通过 RAC1B 诱导 c-Myc 和 Cyclin-D 的增加。在体内，对结直肠癌患者活检的研究表明，与健康对照组相比，所有探索的基因（lncH19、RAC1B、c-Myc 和 Cyclin-D）的水平都更高，从而支持了我们的体外模型。此外，我们在结直肠癌患者中发现了 lncH19 和 RAC1B 之间的正相关关系。最后，我们证明了 lncH19 作为一种穿梭蛋白，将剪接因子 RBFOX2 和 hnRNPM 驱动到 RAC1 上，允许外显子保留和 RAC1B 的表达。本文所示的数据代表了 lncH19 作为一种致癌基因发挥作用的新机制的第一个证据，通过调节剪接，lncH19 促使结直肠癌发生。
