BACKGROUND The NSD family of histone lysine methyltransferases have emerged as important biomarkers that participate in a variety of malignancies. Recent evidence has indicated that somatic dysregulation of the nuclear receptor binding SET domain-containing protein 1 (NSD1) is associated with the tumorigenesis in HCC, suggesting that NSD1 may serve as a prognostic target for this malignant tumor. However, its mechanism in human hepatocellular carcinoma (HCC), the major primary malignant tumor in the human liver, remains unclear. Hence, we investigated how NSD1 regulated HCC progression via regulation of the Wnt/β-catenin signaling pathway. METHODS Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis was performed to identify the expression of NSD1 in HCC cells and clinically obtained tissues. The relationship between NSD1 expression and prognosis was analyzed by Kaplan-Meier survival curve. Further, a NSD1 knockout cell line was constructed by CRISPR/Cas9 genomic editing system, which was investigated in a battery of assays such as HCC cell proliferation, migration and invasion, followed by the investigation into NSD1 regulation on histone H3, Wnt10b and Wnt/β-catenin signaling pathway via ChIP. Finally, a nude mouse xenograft model was conducted in order to assess tumorigenesis affected by NSD1 knockout in vivo. RESULTS NSD1 was overexpressed in HCC tissues and cell lines in association with poor prognosis. Knockout of NSD1 inhibited the proliferation, migration and invasion abilities of HCC cells. CRISPR/Cas9-mediated knockout of NSD1 promoted methylation of H3K27me3 and reduced methylation of H3K36me2, which inhibited Wnt10b expression. The results thereby indicated an inactivation of the Wnt/β-catenin signaling pathway suppressed cell proliferation, migration and invasion in HCC. Moreover, these in vitro findings were reproduced in vivo on tumor xenograft in nude mice. CONCLUSION In conclusion, the study provides evidence that CRISPR/Cas9-mediated NSD1 knockout suppresses HCC cell proliferation and migration via the NSD1/H3/Wnt10b signaling pathway, suggesting that NSD1, H3 and Wnt10b may serve as potential targets for HCC.

中文翻译：

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CRISPR / Cas9介导的NSD1敲除可通过NSD1 / H3 / Wnt10b信号通路抑制肝细胞癌的发展。

背景技术组蛋白赖氨酸甲基转移酶的NSD家族已经作为参与多种恶性肿瘤的重要生物标志物出现。最近的证据表明，核受体结合含SET结构域的蛋白1（NSD1）的体细胞失调与HCC的肿瘤发生有关，这表明NSD1可以作为该恶性肿瘤的预后靶标。但是，其在人肝细胞癌（HCC）中的作用机制尚不清楚，后者是人肝中主要的原发性恶性肿瘤。因此，我们研究了NSD1如何通过Wnt /β-catenin信号通路的调控来调控HCC进程。方法进行逆转录定量聚合酶链反应（RT-qPCR）和蛋白质印迹分析，以鉴定NSD1在肝癌细胞和临床组织中的表达。通过Kaplan-Meier生存曲线分析了NSD1表达与预后之间的关系。此外，通过CRISPR / Cas9基因组编辑系统构建了NSD1基因敲除细胞系，并在一系列检测方法（如HCC细胞增殖，迁移和侵袭）中进行了研究，然后研究了NSD1对组蛋白H3，Wnt10b和Wnt /的调控。通过ChIP的β-catenin信号传导途径。最后，进行裸鼠异种移植模型以评估体内受NSD1敲除的肿瘤发生。结果NSD1在肝癌组织和细胞系中过表达，与预后不良有关。敲除NSD1抑制了HCC细胞的增殖，迁移和侵袭能力。CRISPR / Cas9介导的NSD1敲除促进了H3K27me3的甲基化并降低了H3K36me2的甲基化，抑制了Wnt10b的表达。结果由此表明Wnt /β-连环蛋白信号传导途径的失活抑制了HCC中的细胞增殖，迁移和侵袭。此外，这些体外研究结果在裸鼠体内的肿瘤异种移植物中得以复制。结论总之，该研究提供了证据，证明CRISPR / Cas9介导的NSD1敲除可通过NSD1 / H3 / Wnt10b信号通路抑制HCC细胞增殖和迁移，提示NSD1，H3和Wnt10b可能是HCC的潜在靶标。