CRISPR/Cas9-mediated knockout of PIM3 suppresses tumorigenesis and cancer cell stemness in human hepatoblastoma cells.

Hepatoblastoma remains one of the most difficult childhood tumors to treat and is alarmingly understudied. Over half of patients initially present with locally advanced or metastatic disease and the prognosis for this cohort remains dismal. In addition, many of these children have disease that is resistant to standard therapies and will require novel and targeted therapies to effectively treat or manage their disease. We previously demonstrated that Proviral Insertion site in Maloney murine leukemia virus (PIM) kinases, specifically PIM3, are overexpressed in human hepatoblastoma cells and function to promote tumorigenesis. We aimed to use CRISPR/Cas9 gene editing technology with dual gRNAs to introduce large inactivating deletions in the PIM3 gene and achieve stable PIM3 knockout (KO) in the human hepatoblastoma cell line, HuH6. PIM3 KO of hepatoblastoma cells led to significantly decreased proliferation, viability, and motility, inhibited cell-cycle progression, decreased tumor growth in a xenograft murine model, and increased animal survival. Analysis of RNA sequencing data revealed that PIM3 KO downregulated expression of pro-migratory and pro-invasive genes and upregulated expression of genes involved in apoptosis and differentiation. Furthermore, PIM3 KO decreased hepatoblastoma cancer cell stemness as evidenced by decreased tumorsphere formation, decreased mRNA abundance of stemness markers, and decreased cell surface expression of CD133, a marker of hepatoblastoma stem cell-like cancer cells. Reintroduction of PIM3 into PIM3 KO cells rescued the malignant phenotype. These findings emphasize the role of PIM3 in promoting hepatoblastoma tumorigenesis and provide evidence that targeting PIM3 may offer a novel therapeutic approach for children with hepatoblastoma. Overall design: RNA sequencing of HuH6 WT and PIM3 KO human hepatoblastoma cells.

肝母细胞瘤（Hepatoblastoma）仍是最难治疗的儿童肿瘤之一，相关研究却严重不足。超半数患者初诊时已罹患局部晚期或转移性疾病，该类患者的预后仍不容乐观。此外，此类患儿中不少对标准疗法产生耐药性，需借助新型靶向疗法方可实现有效治疗与病情管控。我们此前的研究证实，莫洛尼鼠白血病病毒前病毒插入位点（Proviral Insertion site in Maloney murine leukemia virus，PIM）激酶，尤其是PIM3，在人肝母细胞瘤细胞中呈过度表达状态，并可促进肿瘤发生。本研究拟采用搭载双向向导RNA（gRNAs）的CRISPR/Cas9基因编辑技术，在PIM3基因中引入大片段失活性缺失，从而在人肝母细胞瘤细胞系HuH6中实现稳定的PIM3基因敲除（knockout，KO）。对肝母细胞瘤细胞进行PIM3敲除后，可显著降低细胞增殖能力、存活率与迁移能力，抑制细胞周期进程，在异种移植小鼠模型中延缓肿瘤生长，并延长实验动物的生存期。对RNA测序（RNA sequencing）数据的分析显示，PIM3敲除会下调促迁移、促侵袭基因的表达，同时上调参与细胞凋亡与分化的基因表达。此外，PIM3敲除还可降低肝母细胞瘤癌细胞的干细胞特性，具体表现为肿瘤球形成能力减弱、干细胞标志物的mRNA丰度降低，以及肝母细胞瘤干细胞样癌细胞表面标志物CD133的表达水平下降。将PIM3重新导入PIM3敲除细胞后，可恢复其恶性表型。上述研究结果明确了PIM3在促进肝母细胞瘤发生发展中的关键作用，并证实靶向PIM3或可为肝母细胞瘤患儿提供全新的治疗策略。整体实验设计：对HuH6野生型（WT）与PIM3敲除型人肝母细胞瘤细胞开展RNA测序。