PDF(Pubmed)
Abstract：
BACKGROUND: Rhabdomyosarcoma (RMS) is a rare malignancy and the most common soft tissue sarcoma in children. Vasculogenic mimicry (VM) is a novel tumor microcirculation model different from traditional tumor angiogenesis, which does not rely on endothelial cells to provide sufficient blood supply for tumor growth. In recent years, VM has been confirmed to be closely associated with tumor progression. However, the ability of RMS to form VM has not yet been reported.
METHODS: Immunohistochemistry, RT-qPCR and western blot were used to test the expression level of SNAI2 and its clinical significance. The biological function in regulating vasculogenic mimicry and malignant progression of SNAI2 was examined both in vitro and in vivo. Mass spectrometry, co-immunohistochemistry, immunofluorescence staining, and ubiquitin assays were performed to explore the regulatory mechanism of SNAI2.
RESULTS: Our study indicated that SNAI2 was abnormally expressed in patients with RMS and RMS cell lines and promoted the proliferation and metastasis of RMS. Through cell tubule formation experiments, nude mice Matrigel plug experiments, and immunohistochemistry (IHC), we confirmed that RMS can form VM and that SNAI2 promotes the formation of VM. Due to SNAI2 is a transcription factor that is not easily drugged, we used Co-IP combined with mass spectrometry to screen for the SNAI2-binding protein USP7 and TRIM21. USP7 depletion inhibited RMS VM formation, proliferation and metastasis by promoting SNAI2 degradation. We further demonstrated that TRIM21 is expressed at low levels in human RMS tissues and inhibits VM in RMS cells. TRIM21 promotes SNAI2 protein degradation through ubiquitination in the RMS. The deubiquitinase USP7 and E3 ligase TRIM21 function in an antagonistic rather than competitive mode and play a key role in controlling the stability of SNAI2 to determine the VM formation and progression of RMS.
CONCLUSIONS: Our findings reveal a previously unknown mechanism by which USP7 and TRIM21 balance the level of SNAI2 ubiquitination, determining RMS vasculogenic mimicry, proliferation, and migration. This new mechanism may provide new targeted therapies to inhibit the development of RMS by restoring TRIM21 expression or inhibiting USP7 expression in RMS patients with high SNAI2 protein levels.
METHODS: Immunohistochemistry, RT-qPCR and western blot were used to test the expression level of SNAI2 and its clinical significance. The biological function in regulating vasculogenic mimicry and malignant progression of SNAI2 was examined both in vitro and in vivo. Mass spectrometry, co-immunohistochemistry, immunofluorescence staining, and ubiquitin assays were performed to explore the regulatory mechanism of SNAI2.
RESULTS: Our study indicated that SNAI2 was abnormally expressed in patients with RMS and RMS cell lines and promoted the proliferation and metastasis of RMS. Through cell tubule formation experiments, nude mice Matrigel plug experiments, and immunohistochemistry (IHC), we confirmed that RMS can form VM and that SNAI2 promotes the formation of VM. Due to SNAI2 is a transcription factor that is not easily drugged, we used Co-IP combined with mass spectrometry to screen for the SNAI2-binding protein USP7 and TRIM21. USP7 depletion inhibited RMS VM formation, proliferation and metastasis by promoting SNAI2 degradation. We further demonstrated that TRIM21 is expressed at low levels in human RMS tissues and inhibits VM in RMS cells. TRIM21 promotes SNAI2 protein degradation through ubiquitination in the RMS. The deubiquitinase USP7 and E3 ligase TRIM21 function in an antagonistic rather than competitive mode and play a key role in controlling the stability of SNAI2 to determine the VM formation and progression of RMS.
CONCLUSIONS: Our findings reveal a previously unknown mechanism by which USP7 and TRIM21 balance the level of SNAI2 ubiquitination, determining RMS vasculogenic mimicry, proliferation, and migration. This new mechanism may provide new targeted therapies to inhibit the development of RMS by restoring TRIM21 expression or inhibiting USP7 expression in RMS patients with high SNAI2 protein levels.
摘要：
背景：横纹肌肉瘤（RMS）是一种罕见的恶性肿瘤，是儿童中最常见的软组织肉瘤。血管生成拟态(VM)是一种不同于传统肿瘤血管生成的新型肿瘤微循环模型,它不依赖内皮细胞为肿瘤生长提供足够的血液供应。近年来,VM已被证实与肿瘤进展密切相关。然而,RMS形成VM的能力尚未报告。
方法：免疫组织化学，RT-qPCR和Westernblot检测SNAI2的表达水平及其临床意义。在体外和体内检查了调节SNAI2的血管生成拟态和恶性进展的生物学功能。质谱,联合免疫组织化学，免疫荧光染色，并进行泛素检测以探讨SNAI2的调控机制。
结果：我们的研究表明SNAI2在RMS患者和RMS细胞系中异常表达，并促进RMS的增殖和转移。通过细胞小管形成实验，裸鼠基质胶塞实验，和免疫组织化学（IHC），我们证实RMS可以形成VM，SNAI2促进VM的形成。由于SNAI2是一种转录因子，不容易被下药，我们使用Co-IP结合质谱来筛选SNAI2结合蛋白USP7和TRIM21.USP7耗尽抑制RMSVM形成，通过促进SNAI2降解进行增殖和转移。我们进一步证明了TRIM21在人RMS组织中以低水平表达并抑制RMS细胞中的VM。TRIM21通过RMS中的泛素化促进SNAI2蛋白降解。去泛素化酶USP7和E3连接酶TRIM21以拮抗而非竞争模式起作用，并在控制SNAI2的稳定性以确定RMS的VM形成和进展中起关键作用。
结论：我们的发现揭示了一种以前未知的机制，USP7和TRIM21平衡SNAI2泛素化水平，确定RMS血管生成拟态，扩散,和移民。这种新机制可能通过恢复TRIM21表达或抑制具有高SNAI2蛋白水平的RMS患者的USP7表达来提供新的靶向治疗以抑制RMS的发展。
方法：免疫组织化学，RT-qPCR和Westernblot检测SNAI2的表达水平及其临床意义。在体外和体内检查了调节SNAI2的血管生成拟态和恶性进展的生物学功能。质谱,联合免疫组织化学，免疫荧光染色，并进行泛素检测以探讨SNAI2的调控机制。
结果：我们的研究表明SNAI2在RMS患者和RMS细胞系中异常表达，并促进RMS的增殖和转移。通过细胞小管形成实验，裸鼠基质胶塞实验，和免疫组织化学（IHC），我们证实RMS可以形成VM，SNAI2促进VM的形成。由于SNAI2是一种转录因子，不容易被下药，我们使用Co-IP结合质谱来筛选SNAI2结合蛋白USP7和TRIM21.USP7耗尽抑制RMSVM形成，通过促进SNAI2降解进行增殖和转移。我们进一步证明了TRIM21在人RMS组织中以低水平表达并抑制RMS细胞中的VM。TRIM21通过RMS中的泛素化促进SNAI2蛋白降解。去泛素化酶USP7和E3连接酶TRIM21以拮抗而非竞争模式起作用，并在控制SNAI2的稳定性以确定RMS的VM形成和进展中起关键作用。
结论：我们的发现揭示了一种以前未知的机制，USP7和TRIM21平衡SNAI2泛素化水平，确定RMS血管生成拟态，扩散,和移民。这种新机制可能通过恢复TRIM21表达或抑制具有高SNAI2蛋白水平的RMS患者的USP7表达来提供新的靶向治疗以抑制RMS的发展。
