Hepatocellular carcinoma (HCC) remains one of the most lethal malignancies. We previously demonstrated that the chromosome 19 microRNA cluster (C19MC) was associated with tumor burden and prognosis in patients with HCC. In the current study, we aim to explore the role of miR-516a-3p—an identical mature microRNA (miRNA) co-spliced by four oncogenic pre-miRNAs of C19MC (i.e., mir-516a-1, mir-516a-2, mir-516b-1, and mir-516b-2)—in HCC. In our cohort of HCC patients, miR-516a-3p was highly expressed in HCC tissues in comparison with adjacent non-tumor tissues. High expression of tumor miR-516a-3p significantly correlated with advanced tumor stages, distinguished high HCC recurrence and mortality, and independently predicted poor prognosis. We further found that miR-516a-3p enhanced the proliferation, migration, and invasiveness of HCC cells in vitro and promoted tumor growth and metastasis in vivo. Among cancer cells, miR-516a-3p could be delivered via exosomes or extracellular vesicles and increased the oncogenic activity of recipient cells. Moreover, we performed comprehensive transcriptomics, proteomics, and metabolomics analysis on the potential mechanism underlying miR-516a-3p-promoted oncogenicity. MixOmic DIABLO analysis showed a close correlation and strong cluster consistency between the proteomics and metabolomics datasets. We further confirmed six proteins (i.e., LMBR1, CHST9, RBM3, SLC7A6, PTGFRN, and NOL12) as the direct targets of miR-516a-3p and as central players in miR-516a-3p-mediated metabolism regulation. The integrated multi-omics and co-enriched pathway analysis showed that miR-516a-3p regulates the metabolic pathways of HCC cells, particularly purine and pyrimidine metabolism. In conclusion, our findings suggest that miR-516a-3p promotes malignant behaviors in HCC cells by regulating cellular metabolism and affecting neighboring cells via the exosome delivery system. Thus, we suggest miR-516a-3p as a novel molecular target for HCC therapy.

肝细胞癌（HCC）仍然是最致命的恶性肿瘤之一。我们之前证明了 19 号染色体 microRNA 簇 (C19MC) 与 HCC 患者的肿瘤负荷和预后相关。在目前的研究中，我们旨在探索 miR-516a-3p 的作用——一种相同的成熟 microRNA (miRNA)，由 C19MC 的四个致癌前 miRNA（即 mir-516a-1、mir-516a-2 、mir-516b-1 和 mir-516b-2)——在 HCC 中。在我们的 HCC 患者队列中，与邻近的非肿瘤组织相比，miR-516a-3p 在 HCC 组织中高度表达。肿瘤 miR-516a-3p 的高表达与晚期肿瘤分期显着相关，可区分高 HCC 复发和死亡率，并独立预测不良预后。我们进一步发现 miR-516a-3p 增强了 HCC 细胞的增殖、迁移和侵袭性在体外并在体内促进肿瘤生长和转移. 在癌细胞中，miR-516a-3p 可以通过外泌体或细胞外囊泡传递，并增加受体细胞的致癌活性。此外，我们对 miR-516a-3p 促进致癌性的潜在机制进行了全面的转录组学、蛋白质组学和代谢组学分析。MixOmic DIABLO 分析显示蛋白质组学和代谢组学数据集之间具有密切的相关性和强的聚类一致性。我们进一步证实了六种蛋白质（即 LMBR1、CHST9、RBM3、SLC7A6、PTGFRN 和 NOL12）作为 miR-516a-3p 的直接靶标和 miR-516a-3p 介导的代谢调节的核心参与者。综合多组学和共富集途径分析表明，miR-516a-3p 调节 HCC 细胞的代谢途径，特别是嘌呤和嘧啶代谢。综上所述，我们的研究结果表明，miR-516a-3p 通过调节细胞代谢和通过外泌体传递系统影响邻近细胞来促进 HCC 细胞的恶性行为。因此，我们建议将 miR-516a-3p 作为 HCC 治疗的新分子靶点。