Luce Amalia, Bocchetti Marco, Cossu Alessia Maria, Tathode Madhura S, Boocock David J, Coveney Clare, Romano Maria Preziosa, De Iesu Maria Roberta, Simeone Ines, Mele Luigi, Vitale Giovanni, Sperlongano Rossella, Misso Gabriella, Verderio Elisabetta A M, Zappavigna Silvia, Caraglia Michele
Department of Precision Medicine, University of Campania "Luigi Vanvitelli", Via L. De Crecchio 7, 80138, Naples, Italy.
John van Geest Cancer Research Centre, Nottingham Trent University, Nottingham, NG11 8NS, UK.
J Transl Med. 2025 Sep 24;23(1):1008. doi: 10.1186/s12967-025-07039-4.
Hepatocellular carcinoma (HCC) remains a significant clinical challenge due to limited diagnostic and therapeutic options. Non-coding RNAs (ncRNAs), such as microRNAs (miRNAs), play key roles in cancer biology. Our previous findings showed that miR-423-5p enhances anti-cancer effects on HCC patients treated with sorafenib by promoting autophagy. Here, we investigated the molecular mechanisms underlying miR-423-5p function through a comprehensive proteomic approach.
We generated an HCC cell line stably overexpressing miR-423-5p via lentiviral transduction. Total proteins were extracted from SNU-387 cells, enzymatically digested into peptides, and subsequently analysed by liquid chromatography-tandem mass spectrometry (LC-MS/M). Raw spectral data were processed and quantified using MaxQuant. Differentially expressed proteins (DEPs) were defined based on fold-change (|log2FC| ≥ 1) and false discovery rate (FDR < 0.05). The full proteomic dataset is available via the ProteomeXchange repository (identifier: PXD064869). Functional enrichment analysis of DEPs were performed using DAVID and Reactome. To assess clinical relevance, predicted and validated miR-423-5p targets were integrated with The Cancer Genome Atlas (TCGA) Liver Hepatocellular Carcinoma (LIHC) dataset using GEPIA platform. Survival analyses were performed using the Kaplan-Meier method.
Proteomic profiling identified 698 DEPs in miR-423-5p-overexpressing cells compared to controls with significant enrichment in metabolic pathways, related to purine/pyrimidine metabolism and gluconeogenesis. Integration with bioinformatic predictions and miRTarBase validation identified 43 DEPs as potential direct targets of miR-423-5p. Among these, seven proteins (ACACA, ANKRD52, DVL3, MCM5, MCM7, RRM2, SPNS1, and SRM) were significantly associated with patient prognosis in the TCGA-LIHC cohort. These targets were downregulated in miR-423-5p-overexpressing cells but upregulated in advanced-stage HCC tissues, suggesting a potential role for miR-423-5p in the regulation of HCC pathogenesis. Stage-specific expression analysis showed increased levels from stage I to III, followed by a decline at stage IV. Notably, we experimentally confirmed miR-423-5p-mediated suppression of MCM7, DVL3, IMPDH1, and SRM (SPEE), supporting their functional involvement in HCC progression.
Overall, our findings support a tumour-suppressive role for miR-423-5p in HCC, mediated by modulation of metabolic pathways and suppression of oncogenic proteins. These results suggest that miR-423-5p and its downstream effectors may serve as promising biomarkers and potential therapeutic targets in HCC.
miR-423-5p acts as a tumor suppressor in HCC by targeting key nodes of pro-tumorigenic signalling. miR-423-5p significantly altered metabolic pathways, including purine/pyrimidine metabolism and gluconeogenesis. Seven miR-423-5p targets correlate with poor prognosis in TCGA-LIHC patients and are downregulated in miR-423-5p overexpressing HCC cells. miR-423-5p over-expression induces a significant downregulation of MCM7, DVL3, IMPDH1, SPEE in HCC cell models. miR-423-5p limits tumor metabolic plasticity, suggesting therapeutic potential.
由于诊断和治疗选择有限，肝细胞癌（HCC）仍然是一个重大的临床挑战。非编码RNA（ncRNAs），如微小RNA（miRNAs），在癌症生物学中发挥关键作用。我们之前的研究结果表明，miR-423-5p通过促进自噬增强了对索拉非尼治疗的HCC患者的抗癌作用。在此，我们通过全面的蛋白质组学方法研究了miR-423-5p功能的分子机制。
我们通过慢病毒转导生成了稳定过表达miR-423-5p的HCC细胞系。从SNU-387细胞中提取总蛋白，酶解成肽段，随后通过液相色谱-串联质谱（LC-MS/MS）进行分析。原始光谱数据使用MaxQuant进行处理和定量。基于倍数变化（|log2FC|≥1）和错误发现率（FDR<0.05）定义差异表达蛋白（DEPs）。完整的蛋白质组数据集可通过ProteomeXchange存储库获取（标识符：PXD064869）。使用DAVID和Reactome对DEPs进行功能富集分析。为了评估临床相关性，使用GEPIA平台将预测和验证的miR-423-5p靶标与癌症基因组图谱（TCGA）肝细胞癌（LIHC）数据集整合。使用Kaplan-Meier方法进行生存分析。
蛋白质组分析显示，与对照组相比，miR-423-5p过表达细胞中有698个DEPs，在与嘌呤/嘧啶代谢和糖异生相关的代谢途径中显著富集。与生物信息学预测和miRTarBase验证相结合，确定了43个DEPs为miR-423-5p的潜在直接靶标。其中，七种蛋白质（ACACA、ANKRD52、DVL3、MCM5、MCM7、RRM2、SPNS1和SRM）与TCGA-LIHC队列中的患者预后显著相关。这些靶标在miR-423-5p过表达细胞中下调，但在晚期HCC组织中上调，表明miR-423-5p在HCC发病机制调控中具有潜在作用。阶段特异性表达分析显示，从I期到III期水平升高，随后在IV期下降。值得注意的是，我们通过实验证实了miR-423-5p介导的对MCM7、DVL3、IMPDH1和SRM（SPEE）的抑制，支持它们在HCC进展中的功能参与。
总体而言，我们的研究结果支持miR-423-5p在HCC中发挥肿瘤抑制作用，通过调节代谢途径和抑制致癌蛋白来实现。这些结果表明，miR-423-5p及其下游效应物可能是HCC中有前景的生物标志物和潜在治疗靶点。
miR-423-5p通过靶向促肿瘤信号传导的关键节点在HCC中发挥肿瘤抑制作用。miR-423-5p显著改变了代谢途径，包括嘌呤/嘧啶代谢和糖异生。七个miR-423-5p靶标与TCGA-LIHC患者的不良预后相关，并且在miR-423-5p过表达的HCC细胞中下调。miR-423-5p过表达在HCC细胞模型中诱导MCM7、DVL3、IMPDH1、SPEE的显著下调。miR-423-5p限制肿瘤代谢可塑性，提示治疗潜力。
