Chen Tong, Chen Jianglong, Zhao Xiuhao, Zhou Jing, Sheng Qingfeng, Zhu Linlin, Lv Zhibao
Department of General Surgery, Shanghai Children's Hospital, Shanghai Jiao Tong University Shanghai 200040, P. R. China.
Am J Cancer Res. 2021 May 15;11(5):1982-2004. eCollection 2021.
Hepatoblastoma (HB) is the most frequent pediatric liver malignancy. However, the treatment outcome for patients with advanced-stage HB remains unsatisfactory. Accumulating evidence indicates that βKlotho (KLB) acts as an oncogene or a tumor-suppressor gene in a context-dependent manner. Despite this, the expression profile and effects of KLB on the growth of HB are still elusive. This study aimed to explore the effect of miR-206/KLB axis on HB growth. The expression of KLB was explored in HB cells (HepG2 and HuH6) and tissues using quantitative polymerase chain reaction (qPCR), Western blot analysis, and immunohistochemistry. Besides, miR-206 expression was determined in HB cells and tissues using qPCR and fluorescence in situ hybridization. The prognostic value of KLB or miR-206 in our patients with HB was investigated using the Kaplan-Meier method. The biological effects of KLB or miR-206 on HB cells were identified . The proliferative effects of KLB on HuH6 cells were also investigated . Moreover, the mechanical signaling of KLB in HB was determined through bioinformatics analysis followed by experimental validation. The results showed a significant upregulation of KLB in HB tissues and cells. Elevated level of KLB was found to be significantly correlated with the aggressive phenotype and poor overall survival for children with HB. The function assay demonstrated that KLB knockdown promoted apoptosis and suppressed the proliferation, migration, and invasion of HB cells. Besides, KLB knockdown inhibited the proliferation of HuH6 cells , while KLB overexpression had the opposite effect. Furthermore, KLB was proved to be the direct target of miR-206. Low level of miR-206 served as an independent risk factor for poor prognosis in children with HB. The overexpression of miR-206 negatively regulated the aggressive biological behaviors of HB cells, which was partially rescued by KLB overexpression. Mechanically, the miR-206/KLB axis played a vital role in HB growth through augmenting the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling. In conclusion, the data demonstrated that the miR-206/KLB axis might serve as an important biomarker/therapeutic target for HB.
肝母细胞瘤（HB）是最常见的儿童肝脏恶性肿瘤。然而，晚期HB患者的治疗效果仍不尽人意。越来越多的证据表明，βKlotho（KLB）在不同情况下可作为癌基因或肿瘤抑制基因。尽管如此，KLB在HB生长中的表达谱和作用仍不清楚。本研究旨在探讨miR-206/KLB轴对HB生长的影响。采用定量聚合酶链反应（qPCR）、蛋白质免疫印迹分析和免疫组织化学方法，检测KLB在HB细胞（HepG2和HuH6）及组织中的表达。此外，采用qPCR和荧光原位杂交技术检测HB细胞及组织中miR-206的表达。用Kaplan-Meier法研究KLB或miR-206对HB患者的预后价值。确定KLB或miR-206对HB细胞的生物学作用。还研究了KLB对HuH6细胞的增殖作用。此外，通过生物信息学分析及实验验证，确定了KLB在HB中的机械信号传导。结果显示，KLB在HB组织和细胞中显著上调。发现KLB水平升高与HB儿童的侵袭性表型及总体生存率低显著相关。功能试验表明，敲低KLB可促进细胞凋亡，并抑制HB细胞的增殖、迁移和侵袭。此外，敲低KLB可抑制HuH6细胞的增殖，而过表达KLB则有相反的作用。此外，KLB被证明是miR-206的直接靶点。低水平的miR-206是HB儿童预后不良的独立危险因素。miR-206的过表达负向调节HB细胞的侵袭性生物学行为，而KLB的过表达可部分逆转这一作用。机制上，miR-206/KLB轴通过增强磷脂酰肌醇3激酶（PI3K）/蛋白激酶B（Akt）/雷帕霉素靶蛋白（mTOR）信号传导，在HB生长中起重要作用。总之，数据表明miR-206/KLB轴可能是HB的重要生物标志物/治疗靶点。
