Division of Gastroenterology, Hepatology and Hepatobiliary Oncology, Aachen Germany; Department of Medicine III, University Hospital RWTH Aachen, Aachen Germany.
Nutrition, Metabolism & Genomics Group, Division of Human Nutrition, Wageningen University, Wageningen, Netherlands.
Gastroenterology. 2018 Dec;155(6):1951-1966.e26. doi: 10.1053/j.gastro.2018.08.032. Epub 2018 Aug 27.
BACKGROUND & AIMS: We performed an integrated analysis to identify microRNAs (miRNAs) and messenger RNAs (mRNAs) with altered expression in liver tumors from 3 mouse models of hepatocellular carcinoma (HCC) and human tumor tissues.
We analyzed miRNA and mRNA expression profiles of liver tissues from mice with diethylnitrosamine-induced hepatocarcinogenesis, conditional expression of lymphotoxin alpha and lymphotoxin beta, or inducible expression of a Myc transgene (Tet-O-Myc mice), as well as male C57BL/6 mice (controls). miRNA mimics were expressed and miRNAs and mRNAs were knocked down in human (Huh7, Hep3B, JHH2) hepatoma cell lines; cells were analyzed for viability, proliferation, apoptosis, migration, and invasion. Cells were grown as xenograft tumors in nude mice and analyzed. We combined in silico target gene prediction with mRNA profiles from all 3 mouse models. We quantified miRNA levels in 146 fresh-frozen tissues from patients (125 HCCs, 17 matched nontumor tissues, and 4 liver samples from patients without cancer) and published human data sets and tested correlations with patient survival times using Kaplan-Meier curves and the log-rank test. Levels of NUSAP1 mRNA were quantified in 237 HCCs and 5 nontumor liver samples using the TaqMan assay.
Levels of the miRNA 193a-5p (MIR193A-5p) were reduced in liver tumors from all 3 mouse tumor models and in human HCC samples, compared with nontumor liver tissues. Expression of a MIR193A-5p mimic in hepatoma cells reduced proliferation, survival, migration, and invasion and their growth as xenograft tumors in nude mice. We found nucleolar and spindle-associated protein 1 (NUSAP1) to be a target of MIR193A-5p; HCC cells and tissues with low levels of MIR193A-5p had increased expression of NUSAP1. Increased levels of NUSAP1 in HCC samples correlated with shorter survival times of patients. Knockdown of NUSAP1 in Huh7 cells reduced proliferation, survival, migration, and growth as xenograft tumors in nude mice. Hydrodynamic tail-vein injections of a small hairpin RNA against NUSAP1 reduced growth of Akt1-Myc-induced tumors in mice.
MIR193A-5p appears to prevent liver tumorigenesis by reducing levels of NUSAP1. Levels of MIR193A-5p are reduced in mouse and human HCC cells and tissues, leading to increased levels of NUSAP1, associated with shorter survival times of patients. Integrated analyses of miRNAs and mRNAs in tumors from mouse models can lead to identification of therapeutic targets in humans. The currently reported miRNA and mRNA profiling data have been submitted to the Gene Expression Omnibus (super-series accession number GSE102418).
我们进行了一项综合分析，以鉴定在 3 种肝癌（HCC）小鼠模型和人类肿瘤组织中肝脏肿瘤中表达改变的 microRNAs（miRNAs）和信使 RNAs（mRNAs）。
我们分析了二乙基亚硝胺诱导的肝癌发生、淋巴毒素 α 和淋巴毒素 β 条件表达或诱导 Myc 转基因（Tet-O-Myc 小鼠）表达的小鼠肝脏组织的 miRNA 和 mRNA 表达谱，以及雄性 C57BL/6 小鼠（对照组）。在人（Huh7、Hep3B、JHH2）肝癌细胞系中表达 miRNA 模拟物并敲低 miRNA 和 mRNAs；分析细胞活力、增殖、凋亡、迁移和侵袭。将细胞作为异种移植肿瘤在裸鼠中生长并进行分析。我们将计算机预测的靶基因与所有 3 种小鼠模型的 mRNA 图谱相结合。我们定量分析了 146 份来自患者（125 例 HCCs、17 例匹配的非肿瘤组织和 4 例无癌症患者的肝脏样本）的新鲜冷冻组织中的 miRNA 水平，并使用 Kaplan-Meier 曲线和对数秩检验测试了与患者生存时间的相关性。使用 TaqMan 测定法定量分析了 237 例 HCC 和 5 例非肿瘤性肝组织中的 NUSAP1 mRNA 水平。
与非肿瘤性肝组织相比，所有 3 种小鼠肿瘤模型和人类 HCC 样本中的 miRNA 193a-5p（MIR193A-5p）水平降低。在肝癌细胞中表达 MIR193A-5p 模拟物可降低增殖、存活、迁移和侵袭以及其在裸鼠中的异种移植肿瘤生长。我们发现核仁纺锤体相关蛋白 1（NUSAP1）是 MIR193A-5p 的靶标；MIR193A-5p 水平较低的 HCC 细胞和组织中 NUSAP1 的表达增加。HCC 样本中 NUSAP1 水平的增加与患者生存时间的缩短相关。在 Huh7 细胞中敲低 NUSAP1 可降低增殖、存活、迁移和生长作为裸鼠中的异种移植肿瘤。尾静脉注射针对 NUSAP1 的短发夹 RNA 可减少 Akt1-Myc 诱导的肿瘤在小鼠中的生长。
MIR193A-5p 通过降低 NUSAP1 水平似乎可预防肝肿瘤发生。MIR193A-5p 在小鼠和人类 HCC 细胞和组织中的水平降低，导致 NUSAP1 水平升高，与患者生存时间缩短相关。对小鼠模型肿瘤中 miRNA 和 mRNAs 的综合分析可导致鉴定人类的治疗靶点。目前报道的 miRNA 和 mRNA 分析数据已提交给基因表达综合数据库（超级系列注册号 GSE102418）。
