CRISPR/Cas9 Engineering of Adult Mouse Liver Demonstrates That the Dnajb1-Prkaca Gene Fusion Is Sufficient to Induce Tumors Resembling Fibrolamellar Hepatocellular Carcinoma.
CRISPR/Cas9 Engineering of Adult Mouse Liver Demonstrates That the Dnajb1-Prkaca Gene Fusion Is Sufficient to Induce Tumors Resembling Fibrolamellar Hepatocellular Carcinoma.
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DOI：
10.1053/j.gastro.2017.09.008
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发表时间：
2017-12
期刊：
影响因子：
29.4
通讯作者：
Frödin M
中科院分区：
文献类型：
--
作者：
Engelholm LH;Riaz A;Serra D;Dagnæs-Hansen F;Johansen JV;Santoni-Rugiu E;Hansen SH;Niola F;Frödin M
纤维板层状肝细胞癌(FL-HCC)是一种主要影响年轻人的原发性肝癌，没有潜在的肝脏疾病。在FL-HCC患者中，反复发现19号染色体上一个400kb的体细胞缺失，该缺失将DNAJ热休克蛋白家族(Hsp40)成员B1基因(DNAJB1)的一部分融合到蛋白激酶cAMP激活的催化亚单位α基因(PRKACA)中。然而，DNAJB1-PRKACA基因融合尚未被证明能诱导肝肿瘤的发生。我们使用CRISPR/Cas9技术删除小鼠8号染色体上的同线区域，以创建DNAJB1-PRKACA融合，并监测小鼠的肝脏肿瘤发展。我们将DNAJB1的外显子1和PRKACA的外显子2并列的CRISPR/Cas9载体，通过尾静脉注射到8周龄雌性FVB/N小鼠的肝脏，构建了与FL-HCC相关的DNAJB1-PRKACA基因融合或对照Cas9载体。这些小鼠没有任何其他的基因工程改变，也没有暴露在肝脏毒素或致癌物中。产后14个月取肝组织，用聚合酶链式反应检测DNAJB1-PRKACA融合，组织学、免疫组织化学、RNA测序、全外显子测序。在给予载体的15只小鼠中，有12只小鼠的肝脏诱导DNAJB1-PRKACA基因融合，但给予对照载体的11只小鼠中，没有一只发生肿瘤。该肿瘤含有DNAJB1-PRKACA基因融合基因，具有人FL-HC的组织学和细胞学特征：大的多角形细胞，胞浆呈颗粒状、嗜酸性和线粒体丰富，核仁突出，有肝细胞和胆管细胞的标志。在比较小鼠肿瘤和非肿瘤肝细胞的基因表达水平时，我们发现了与人类FL-HCC相似的变化，其中包括影响细胞周期和有丝分裂调控的基因。对DNAJB1-PRKACA融合诱导的小鼠肿瘤的基因组分析显示，与人类FL-HCC中观察到的那样，与肝癌相关的基因缺乏突变。利用CRISPR/Cas9技术，我们发现在野生型小鼠中产生的DNAJB1-PRKACA融合基因足以启动具有许多人类FL-HCC特征的肿瘤的形成。阻断DNAJB1-PRKACA的策略可能被开发为这种形式的肝癌的治疗方法。
Fibrolamellar hepatocellular carcinoma (FL-HCC) is a primary liver cancer that predominantly affects young adults with no underlying liver disease. A somatic, 400 Kb deletion on chromosome 19 that fuses part of the DnaJ heat shock protein family (Hsp40) member B1 gene (DNAJB1) to the protein kinase cAMP-activated catalytic subunit alpha gene (PRKACA) has been repeatedly identified in patients with FL-HCC. However, the DNAJB1–PRKACA gene fusion has not been shown to induce liver tumorigenesis. We used the CRISPR/Cas9 technique to delete in mice the syntenic region on chromosome 8 to create a Dnajb1–Prkaca fusion and monitored the mice for liver tumor development. We delivered CRISPR/Cas9 vectors designed to juxtapose exon 1 of Dnajb1 with exon 2 of Prkaca to create the Dnajb1–Prkaca gene fusion associated with FL-HCC, or control Cas9 vector, via hydrodynamic tail vein injection to livers of 8 week-old female FVB/N mice. These mice did not have any other engineered genetic alterations and were not exposed to liver toxins or carcinogens. Liver tissues were collected 14 months after delivery; genomic DNA was analyzed by PCR to detect the Dnajb1–Prkaca fusion, and tissues were characterized by histology, immunohistochemistry, RNA sequencing, and whole-exome sequencing. Livers from 12 of the 15 mice given the vectors to induce the Dnajb1–Prkaca gene fusion, but none of the 11 mice given the control vector, developed neoplasms. The tumors contained the Dnajb1–Prkaca gene fusion and had histologic and cytologic features of human FL-HCCs: large polygonal cells with granular, eosinophilic, and mitochondria-rich cytoplasm, prominent nucleoli, and markers of hepatocytes and cholangiocytes. In comparing expression levels of genes between the mouse tumor and non-tumor liver cells, we identified changes similar to those detected in human FL-HCC, which included genes that affect cell cycle and mitosis regulation. Genomic analysis of mouse neoplasms induced by the Dnajb1–Prkaca fusion revealed a lack of mutations in genes commonly associated with liver cancers, as observed in human FL-HCC. Using CRISPR/Cas9 technology, we found generation of the Dnajb1–Prkaca fusion gene in wild-type mice to be sufficient to initiate formation of tumors that have many features of human FL-HCC. Strategies to block DNAJB1–PRKACA might be developed as therapeutics for this form of liver cancer.
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影响因子：
4.7
作者：
Dagnæs-Hansen, F;Holst, HU;Jensen, TG
通讯作者：
Jensen, TG
影响因子：
4.6
作者：
Dinh TA;Vitucci EC;Wauthier E;Graham RP;Pitman WA;Oikawa T;Chen M;Silva GO;Greene KG;Torbenson MS;Reid LM;Sethupathy P
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Sethupathy P
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作者：
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Matsuda, Koichi
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25.7
作者：
HODGSON, HJF
通讯作者：
HODGSON, HJF
影响因子：
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作者：
Kakar, S;Burgart, LJ;Ferrell, LD
通讯作者：
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