Maimónides Institute of Biomedical Research of Córdoba (IMIBIC), 14004, Córdoba, Spain.
Department of Cell Biology, Physiology and Immunology, University of Córdoba, 14004, Córdoba, Spain.
Exp Mol Med. 2023 Jan;55(1):132-142. doi: 10.1038/s12276-022-00917-7. Epub 2023 Jan 6.
Hepatocellular carcinoma (HCC) pathogenesis is associated with alterations in splicing machinery components (spliceosome and splicing factors) and aberrant expression of oncogenic splice variants. We aimed to analyze the expression and potential role of the spliceosome component PRPF8 (pre-mRNA processing factor 8) in HCC. PRPF8 expression (mRNA/protein) was analyzed in a retrospective cohort of HCC patients (n = 172 HCC and nontumor tissues) and validated in two in silico cohorts (TCGA and CPTAC). PRPF8 expression was silenced in liver cancer cell lines and in xenograft tumors to understand the functional and mechanistic consequences. In silico RNAseq and CLIPseq data were also analyzed. Our results indicate that PRPF8 is overexpressed in HCC and associated with increased tumor aggressiveness (patient survival, etc.), expression of HCC-related splice variants, and modulation of critical genes implicated in cancer-related pathways. PRPF8 silencing ameliorated aggressiveness in vitro and decreased tumor growth in vivo. Analysis of in silico CLIPseq data in HepG2 cells demonstrated that PRPF8 binds preferentially to exons of protein-coding genes, and RNAseq analysis showed that PRPF8 silencing alters splicing events in multiple genes. Integrated and in vitro analyses revealed that PRPF8 silencing modulates fibronectin (FN1) splicing, promoting the exclusion of exon 40.2, which is paramount for binding to integrins. Consistent with this finding, PRPF8 silencing reduced FAK/AKT phosphorylation and blunted stress fiber formation. Indeed, HepG2 and Hep3B cells exhibited a lower invasive capacity in membranes treated with conditioned medium from PRPF8-silenced cells compared to medium from scramble-treated cells. This study demonstrates that PRPF8 is overexpressed and associated with aggressiveness in HCC and plays important roles in hepatocarcinogenesis by altering FN1 splicing, FAK/AKT activation and stress fiber formation.
肝细胞癌 (HCC) 的发病机制与剪接机制组件 (剪接体和剪接因子) 的改变以及致癌剪接变异体的异常表达有关。我们旨在分析剪接体成分 PRPF8 (mRNA 前体处理因子 8) 在 HCC 中的表达及其潜在作用。在一个回顾性 HCC 患者队列 (n=172 例 HCC 和非肿瘤组织) 中分析了 PRPF8 的表达 (mRNA/蛋白)，并在两个计算队列 (TCGA 和 CPTAC) 中进行了验证。在肝癌细胞系和异种移植肿瘤中沉默 PRPF8 表达，以了解其功能和机制后果。还分析了计算 RNAseq 和 CLIPseq 数据。我们的结果表明，PRPF8 在 HCC 中过度表达，与肿瘤侵袭性增加 (患者生存等)、HCC 相关剪接变异体的表达以及参与癌症相关途径的关键基因的调节有关。PRPF8 沉默可改善体外侵袭性并减少体内肿瘤生长。在 HepG2 细胞中分析计算 CLIPseq 数据表明，PRPF8 优先结合蛋白质编码基因的外显子，RNAseq 分析表明，PRPF8 沉默会改变多个基因的剪接事件。整合和体外分析表明，PRPF8 沉默可调节纤连蛋白 (FN1) 的剪接，促进外显子 40.2 的排除，这对于与整合素结合至关重要。与这一发现一致的是，PRPF8 沉默减少了 FAK/AKT 磷酸化并减弱了应力纤维的形成。事实上，与用对照细胞处理的条件培养基相比，PRPF8 沉默细胞的 HepG2 和 Hep3B 细胞在膜中的侵袭能力较低。这项研究表明，PRPF8 在 HCC 中过度表达并与侵袭性相关，通过改变 FN1 剪接、FAK/AKT 激活和应力纤维形成在肝癌发生中发挥重要作用。
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