Mohammed Mohammed Ahmed, Soffar Ahmed Abdelmagied, Yousef Amany I, Salama Mohammed, Ibrahim Fawziya A R, El-Sewedy Tarek, Elmetwalli Alaa
Department of Applied Medical Chemistry, Medical Research Institute, Alexandria University, Alexandria, Egypt.
Department of Zoology, Faculty of Sciences, Alexandria University, Alexandria, Egypt.
Funct Integr Genomics. 2025 Jul 11;25(1):153. doi: 10.1007/s10142-025-01663-5.
Colorectal cancer (CRC) remains a leading cause of cancer-related mortality worldwide, with tumor progression often driven by dysregulated oncogenic pathways. USP6NL, a known regulator of endocytic trafficking, has recently been implicated in tumorigenesis. However, its precise role in CRC remains unclear, and more studies are still needed to deepen our understanding of underlying mechanisms implicated in its oncogenic role. Therefore, silencing USP6NL could provide a novel therapeutic strategy by concurrently disrupting several oncogenic mechanisms, creating a new avenue for CRC management, particularly in patients who develop resistance against conventional therapies. This study investigates the impact of USP6NL knockdown on CRC cell morphology, proliferation, apoptosis, migration, angiogenesis, and metabolic adaptation, providing mechanistic insights into its oncogenic functions.
HCT116 colorectal cancer cells were transfected with USP6NL-specific siRNA. Immunocytochemistry was used to confirm successful silencing, functional assays were performed to assess changes in cell morphology using phase-contrast and scanning electron microscopy, and colony formation and wound healing assays were performed to assess cell clonogenic capacity and migration, respectively, in addition to apoptosis assay via flow cytometry, and RT²-Profiler PCR array to measure variation in gene expression of 84 cancer-related genes. Statistical analyses were performed to evaluate significant differences between control and USP6NL-silenced groups.
USP6NL depletion led to profound morphological changes, including membrane blebbing, cell shrinkage, and loss of adhesion, reflecting late apoptotic features of cells. These findings were further supported by flow cytometry, which confirmed increased apoptosis, with a higher proportion of late apoptotic cells (20.99% in USP6NL knockdown vs. 2.69% in control, p = 0.042). Colony formation assays revealed a significant reduction in the clonogenic potential, suggesting a critical role of USP6NL in promoting CRC cell proliferation (p ≤ 0.05). The wound healing assay demonstrated impaired migration in USP6NL-silenced cells, with a marked delay in wound closure (p = 0.0201 at 48 h). Gene expression analysis revealed a significant downregulation of VEGFC (-8.62-fold) and ANGPT2 (-4.03-fold), impairing angiogenesis and suppressing FOXC2, SNAI1, and SNAI2, indicating EMT inhibition. Additionally, CASP9, APAF1, and BCL2L11 were upregulated, confirming the activation of intrinsic apoptosis, while metabolic regulators HIF1A and LDHA were downregulated, suggesting impaired tumor hypoxic adaptation.
This study establishes USP6NL as a key modulator of CRC progression, regulating proliferation, apoptosis, migration, angiogenesis, and metabolic pathways. The loss of USP6NL leads to EMT suppression, apoptosis induction, and reduced tumor cell viability, positioning it as a potential therapeutic target in colorectal cancer. Further investigations are warranted to explore USP6NL's interactions in oncogenic signaling networks and its feasibility as a target for CRC therapy. It could serve as a promising therapeutic target in colorectal cancer, potentially enhancing tumor cell death and limiting metastasis. Targeting USP6NL could also provide a novel approach in combination with existing therapies, improving treatment efficacy and reducing side effects.
结直肠癌（CRC）仍是全球癌症相关死亡的主要原因之一，肿瘤进展通常由失调的致癌途径驱动。USP6NL是一种已知的内吞运输调节因子，最近被认为与肿瘤发生有关。然而，其在结直肠癌中的精确作用仍不清楚，仍需要更多研究来加深我们对其致癌作用潜在机制的理解。因此，沉默USP6NL可能通过同时破坏多种致癌机制提供一种新的治疗策略，为结直肠癌的治疗开辟一条新途径，特别是对于那些对传统疗法产生耐药性的患者。本研究调查了USP6NL基因敲低对结直肠癌细胞形态、增殖、凋亡、迁移、血管生成和代谢适应的影响，为其致癌功能提供了机制上的见解。
用USP6NL特异性小干扰RNA转染HCT116结直肠癌细胞。采用免疫细胞化学法确认成功沉默，通过相差显微镜和扫描电子显微镜进行功能测定以评估细胞形态变化，进行集落形成和伤口愈合试验分别评估细胞克隆形成能力和迁移能力，此外通过流式细胞术进行凋亡检测，并通过RT²- Profiler PCR芯片测量84个癌症相关基因的基因表达变化。进行统计分析以评估对照组和USP6NL沉默组之间的显著差异。
USP6NL基因缺失导致细胞形态发生显著变化，包括细胞膜起泡、细胞收缩和黏附丧失，反映出细胞的晚期凋亡特征。流式细胞术进一步支持了这些发现，证实凋亡增加，晚期凋亡细胞比例更高（USP6NL基因敲低组为20.99%，对照组为2.69%，p = 0.042）。集落形成试验显示克隆形成潜力显著降低，表明USP6NL在促进结直肠癌细胞增殖中起关键作用（p≤0.05）。伤口愈合试验表明USP6NL沉默细胞的迁移受损，伤口闭合明显延迟（48小时时p = 0.0201）。基因表达分析显示VEGFC（-8.62倍）和ANGPT2（-4.03倍）显著下调，损害血管生成并抑制FOXC2、SNAI1和SNAI2，表明上皮-间质转化（EMT）受到抑制。此外，CASP9、APAF1和BCL2L11上调，证实内源性凋亡被激活，而代谢调节因子HIF1A和LDHA下调，表明肿瘤缺氧适应受损。
本研究确定USP6NL是结直肠癌进展的关键调节因子，调节增殖、凋亡、迁移、血管生成和代谢途径。USP6NL的缺失导致EMT抑制、凋亡诱导和肿瘤细胞活力降低，使其成为结直肠癌潜在的治疗靶点。有必要进一步研究以探索USP6NL在致癌信号网络中的相互作用及其作为结直肠癌治疗靶点的可行性。它可能是结直肠癌中有前景的治疗靶点，有可能增强肿瘤细胞死亡并限制转移。靶向USP6NL还可为联合现有疗法提供新方法，提高治疗效果并减少副作用。
