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Abstract：
Circ-CCT2 (hsa_circ_0000418) is a novel circular RNA that stems from the CCT2 gene. However, the expression of circ-CCT2 and its roles in hepatoblastoma are unknown. Our study aims to study the circ-CCT2 roles in hepatoblastoma development.
Hepatoblastoma specimens were collected for examining the expression of circ-CCT2, TAF15, and PTBP1. CCK-8 and colony formation assays were applied for cell proliferation analysis. Migratory and invasive capacities were evaluated through wound healing and Transwell assays. The interaction between circ-CCT2, TAF15, and PTBP1 was validated by fluorescence in situ hybridization, RNA pull-down, and RNA immunoprecipitation. SKL2001 was used as an agonist of the Wnt/β-catenin pathway. A subcutaneous mouse model of hepatoblastoma was established for examining the function of circ-CCT2 in hepatoblastoma in vivo.
Circ-CCT2 was significantly up-regulated in hepatoblastoma. Overexpression of circ-CCT2 activated Wnt/β-catenin signaling and promoted hepatoblastoma progression, whereas knockdown of circ-CCT2 exerted opposite effects. Moreover, both TAF15 and PTBP1 were up-regulated in hepatoblastoma tissues and cells. TAF15 was positively correlated with the expression of circ-CCT2 and PTBP1 in hepatoblastoma. Furthermore, circ-CCT2 recruited and up-regulated TAF15 protein to stabilize PTBP1 mRNA and trigger Wnt/β-catenin signaling in hepatoblastoma. Overexpression of TAF15 or PTBP1 reversed knockdown of circ-CCT2-mediated suppression of hepatoblastoma progression. SKL2001-mediated activation of Wnt/β-catenin signaling reversed the anti-tumor effects of silencing of circ-CCT2, TAF15, or PTBP1.
Circ-CCT2 stabilizes PTBP1 mRNA and activates Wnt/β-catenin signaling through recruiting and up-regulating TAF15 protein, thus promoting hepatoblastoma progression. Our findings deepen the understanding of hepatoblastoma pathogenesis and suggest potential therapeutic targets.
Hepatoblastoma specimens were collected for examining the expression of circ-CCT2, TAF15, and PTBP1. CCK-8 and colony formation assays were applied for cell proliferation analysis. Migratory and invasive capacities were evaluated through wound healing and Transwell assays. The interaction between circ-CCT2, TAF15, and PTBP1 was validated by fluorescence in situ hybridization, RNA pull-down, and RNA immunoprecipitation. SKL2001 was used as an agonist of the Wnt/β-catenin pathway. A subcutaneous mouse model of hepatoblastoma was established for examining the function of circ-CCT2 in hepatoblastoma in vivo.
Circ-CCT2 was significantly up-regulated in hepatoblastoma. Overexpression of circ-CCT2 activated Wnt/β-catenin signaling and promoted hepatoblastoma progression, whereas knockdown of circ-CCT2 exerted opposite effects. Moreover, both TAF15 and PTBP1 were up-regulated in hepatoblastoma tissues and cells. TAF15 was positively correlated with the expression of circ-CCT2 and PTBP1 in hepatoblastoma. Furthermore, circ-CCT2 recruited and up-regulated TAF15 protein to stabilize PTBP1 mRNA and trigger Wnt/β-catenin signaling in hepatoblastoma. Overexpression of TAF15 or PTBP1 reversed knockdown of circ-CCT2-mediated suppression of hepatoblastoma progression. SKL2001-mediated activation of Wnt/β-catenin signaling reversed the anti-tumor effects of silencing of circ-CCT2, TAF15, or PTBP1.
Circ-CCT2 stabilizes PTBP1 mRNA and activates Wnt/β-catenin signaling through recruiting and up-regulating TAF15 protein, thus promoting hepatoblastoma progression. Our findings deepen the understanding of hepatoblastoma pathogenesis and suggest potential therapeutic targets.
摘要：
目的：Circ-CCT2（hsa_circ_0000418）是一种源于CCT2基因的新型环状RNA。然而,circ-CCT2的表达及其在肝母细胞瘤中的作用尚不清楚。我们的研究旨在研究circ-CCT2在肝母细胞瘤发展中的作用。
方法：收集肝母细胞瘤标本检测circ-CCT2、TAF15和PTBP1的表达。将CCK-8和集落形成测定用于细胞增殖分析。通过伤口愈合和transwell测定评估迁移和侵入能力。通过FISH验证了Circ-CCT2、TAF15和PTBP1之间的相互作用，RNA下拉和RIP。SKL2001用作Wnt/β-连环蛋白途径的激动剂。建立肝母细胞瘤的皮下小鼠模型，以检测circ-CCT2在体内肝母细胞瘤中的功能。
结果：Circ-CCT2在肝母细胞瘤中显著上调。circ-CCT2过表达激活Wnt/β-catenin信号并促进肝母细胞瘤进展,而circ-CCT2的敲低产生相反的效果。此外，TAF15和PTBP1在肝母细胞瘤组织和细胞中均上调.TAF15与circ-CCT2和PTBP1在肝母细胞瘤中的表达呈正相关。此外，circ-CCT2在肝母细胞瘤中募集并上调TAF15蛋白以稳定PTBP1mRNA并触发Wnt/β-catenin信号传导。TAF15或PTBP1的过表达逆转了circ-CCT2介导的对肝母细胞瘤进展的抑制。SKL2001介导的Wnt/β-catenin信号的激活逆转了circ-CCT2，TAF15或PTBP1沉默的抗肿瘤作用。
结论：Circ-CCT2通过募集和上调TAF15蛋白来稳定PTBP1mRNA并激活Wnt/β-catenin信号，从而促进肝母细胞瘤的进展。我们的发现加深了对肝母细胞瘤发病机制的理解，并提出了潜在的治疗靶点。
方法：收集肝母细胞瘤标本检测circ-CCT2、TAF15和PTBP1的表达。将CCK-8和集落形成测定用于细胞增殖分析。通过伤口愈合和transwell测定评估迁移和侵入能力。通过FISH验证了Circ-CCT2、TAF15和PTBP1之间的相互作用，RNA下拉和RIP。SKL2001用作Wnt/β-连环蛋白途径的激动剂。建立肝母细胞瘤的皮下小鼠模型，以检测circ-CCT2在体内肝母细胞瘤中的功能。
结果：Circ-CCT2在肝母细胞瘤中显著上调。circ-CCT2过表达激活Wnt/β-catenin信号并促进肝母细胞瘤进展,而circ-CCT2的敲低产生相反的效果。此外，TAF15和PTBP1在肝母细胞瘤组织和细胞中均上调.TAF15与circ-CCT2和PTBP1在肝母细胞瘤中的表达呈正相关。此外，circ-CCT2在肝母细胞瘤中募集并上调TAF15蛋白以稳定PTBP1mRNA并触发Wnt/β-catenin信号传导。TAF15或PTBP1的过表达逆转了circ-CCT2介导的对肝母细胞瘤进展的抑制。SKL2001介导的Wnt/β-catenin信号的激活逆转了circ-CCT2，TAF15或PTBP1沉默的抗肿瘤作用。
结论：Circ-CCT2通过募集和上调TAF15蛋白来稳定PTBP1mRNA并激活Wnt/β-catenin信号，从而促进肝母细胞瘤的进展。我们的发现加深了对肝母细胞瘤发病机制的理解，并提出了潜在的治疗靶点。
