Curriculum in Genetics and Molecular Biology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York.
Department of Medicine, School of Medicine, Duke University, Durham, North Carolina.
Cell Mol Gastroenterol Hepatol. 2019;7(4):803-817. doi: 10.1016/j.jcmgh.2019.01.008. Epub 2019 Feb 11.
BACKGROUND & AIMS: Fibrolamellar carcinoma (FLC) is a rare liver cancer that primarily affects adolescents and young adults. It is characterized by a heterozygous approximately 400-kb deletion on chromosome 19 that results in a unique fusion between DnaJ heat shock protein family member B1 (DNAJB1) and the alpha catalytic subunit of protein kinase A (PRKACA). The role of microRNAs (miRNAs) in FLC remains unclear. We identified dysregulated miRNAs in FLC and investigated whether dysregulation of 1 key miRNA contributes to FLC pathogenesis.
We analyzed small RNA sequencing (smRNA-seq) data from The Cancer Genome Atlas to identify dysregulated miRNAs in primary FLC tumors and validated the findings in 3 independent FLC cohorts. smRNA-seq also was performed on a FLC patient-derived xenograft model as well as purified cell populations of the liver to determine whether key miRNA changes were tumor cell-intrinsic. We then used clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (Cas9) technology and transposon-mediated gene transfer in mice to determine if the presence of DNAJB1-PRKACA is sufficient to suppress miR-375 expression. Finally, we established a new FLC cell line and performed colony formation and scratch wound assays to determine the functional consequences of miR-375 overexpression.
We identified miR-375 as the most dysregulated miRNA in primary FLC tumors (27-fold down-regulation; P = .009). miR-375 expression also was decreased significantly in a FLC patient-derived xenograft model compared to 4 different cell populations of the liver. Introduction of DNAJB1-PRKACA by clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 engineering and transposon-mediated somatic gene transfer in mice was sufficient to induce significant loss of miR-375 expression (P < .05). Overexpression of miR-375 in FLC cells inhibited Hippo signaling pathway proteins, including yes-associated protein 1 and connective tissue growth factor, and suppressed cell proliferation and migration (P < .05).
We identified miR-375 as the most down-regulated miRNA in FLC tumors and showed that overexpression of miR-375 mitigated tumor cell growth and invasive potential. These findings open a potentially new molecular therapeutic approach. Further studies are necessary to determine how DNAJB1-PRKACA suppresses miR-375 expression and whether miR-375 has additional important targets in this tumor. Transcript profiling: GEO accession numbers: GSE114974 and GSE125602.
纤维板层样肝癌（FLC）是一种罕见的肝癌，主要影响青少年和年轻成年人。它的特征是染色体 19 上存在大约 400kb 的杂合缺失，导致 DnaJ 热休克蛋白家族成员 B1（DNAJB1）和蛋白激酶 A 的α催化亚基（PRKACA）之间的独特融合。microRNAs（miRNAs）在 FLC 中的作用尚不清楚。我们鉴定了 FLC 中失调的 miRNAs，并研究了关键 miRNA 的失调是否有助于 FLC 的发病机制。
我们分析了来自癌症基因组图谱的小 RNA 测序（smRNA-seq）数据，以鉴定原发性 FLC 肿瘤中失调的 miRNAs，并在 3 个独立的 FLC 队列中验证了这些发现。smRNA-seq 还在 FLC 患者来源的异种移植模型以及纯化的肝细胞群中进行，以确定关键 miRNA 变化是否为肿瘤细胞内在的。然后，我们使用成簇规律间隔短回文重复/CRISPR 相关蛋白 9（Cas9）技术和转座子介导的基因转移在小鼠中确定 DNAJB1-PRKACA 的存在是否足以抑制 miR-375 的表达。最后，我们建立了一个新的 FLC 细胞系，并进行了集落形成和划痕伤口分析，以确定 miR-375 过表达的功能后果。
我们将 miR-375 鉴定为原发性 FLC 肿瘤中最失调的 miRNA（下调 27 倍；P =.009）。与 4 种不同的肝细胞群相比，miR-375 在 FLC 患者来源的异种移植模型中的表达也显著降低。在小鼠中通过成簇规律间隔短回文重复/CRISPR 相关蛋白 9 工程和转座子介导的体细胞基因转移引入 DNAJB1-PRKACA 足以诱导 miR-375 表达的显著丧失（P <.05）。在 FLC 细胞中过表达 miR-375 抑制 Hippo 信号通路蛋白，包括 yes 相关蛋白 1 和结缔组织生长因子，并抑制细胞增殖和迁移（P <.05）。
我们将 miR-375 鉴定为 FLC 肿瘤中下调最明显的 miRNA，并表明 miR-375 的过表达减轻了肿瘤细胞的生长和侵袭潜力。这些发现开辟了一种潜在的新的分子治疗方法。进一步的研究有必要确定 DNAJB1-PRKACA 如何抑制 miR-375 的表达，以及 miR-375 在这种肿瘤中是否还有其他重要的靶标。转录谱分析：GEO 注册号：GSE114974 和 GSE125602。
