miR-23a ~ 27a ~ 24-2 簇在包括肝细胞癌 (HCC) 在内的多种癌症中普遍上调，引发了对其三种成熟 miRNA 的具体功能及其整合功能的疑问。利用CRISPR敲除（KO）、CRISPR干扰（CRISPRi）和CRISPR激活（CRISPRa）技术，我们建立了受控的内源性miR-23a ~ 27 ~ a24-2细胞模型，以揭示它们在HCC中的作用和信号通路。 miR-23a KO 和 miR-27a KO 均在体外和体内表现出细胞生长减少，揭示了整合的致癌功能。功能分析表明，细胞周期停滞，特别是在 G2/M 期，是通过 CDK1/细胞周期蛋白 B 激活的下调实现的。高通量 RNA-seq 与 miRNA 靶标预测相结合，揭示了 miR-23a/miR-27a 调控的基因网络，并通过多种技术进行了验证。虽然 miR-23a 和 miR-27a 在细胞迁移和间质-上皮转化中表现出相反的作用，但整合的 CRISPRi/a 分析表明 miR-23a ~ 27a ~ 24-2 簇在细胞迁移中具有致癌作用。这种参与可能涵盖两个信号轴：HCC 细胞中的 miR-23a-BMPR2 和 miR-27a-TMEM170B。总之，我们的 CRISPRi/a 研究为理解内源 miRNA 簇的综合作用和潜在机制提供了一个有价值的工具，为 miRNA 靶向治疗干预的有希望的方向铺平了道路。© 2024。作者。

The miR-23a ~ 27a ~ 24-2 cluster, commonly upregulated in diverse cancers, including hepatocellular carcinoma (HCC), raises questions about the specific functions of its three mature miRNAs and their integrated function. Utilizing CRISPR knockout (KO), CRISPR interference (CRISPRi), and CRISPR activation (CRISPRa) technologies, we established controlled endogenous miR-23a ~ 27 ~ a24-2 cell models to unravel their roles and signaling pathways in HCC. Both miR-23a KO and miR-27a KO displayed reduced cell growth in vitro and in vivo, revealing an integrated oncogenic function. Functional analysis indicated cell cycle arrest, particularly at the G2/M phase, through the downregulation of CDK1/cyclin B activation. High-throughput RNA-seq, combined with miRNA target prediction, unveiled the miR-23a/miR-27a-regulated gene network, validated through diverse technologies. While miR-23a and miR-27a exhibited opposing roles in cell migration and mesenchymal-epithelial transition, an integrated CRISPRi/a analysis suggested an oncogenic role of the miR-23a ~ 27a ~ 24-2 cluster in cell migration. This involvement potentially encompasses two signaling axes: miR-23a-BMPR2 and miR-27a-TMEM170B in HCC cells. In conclusion, our CRISPRi/a study provides a valuable tool for comprehending the integrated roles and underlying mechanisms of endogenous miRNA clusters, paving the way for promising directions in miRNA-targeted therapy interventions.© 2024. The Author(s).