Data_Sheet_3_Generation and Validation of miR-100 Hepatocyte-Specific Knock-Out Mice.PDF

Background: Inactivation of microRNA-100 (miR-100) is involved in hepatocellular carcinoma (HCC) and miR-100 behaves as a tumor suppressor. To understand miR-100 function in HCC genesis and development in vivo, we developed hepatocyte-specific miR-100 deficient mice.
Methods: Mice homozygous for floxed miR-100 allele that carried the Alb-Cre transgene (miR-100flox/floxAlb -Cre+) were developed by mating miR-100flox/flox mice with Alb-Cre+/+mice. The mice tails DNA were genotyped using the primers for LoxP sites and Cre recombinase, respectively. The specific deletion of miR-100 in the livers was verified by quantitative Real-time PCR (qRT-PCR). HE-staining was performed for histology analysis. Liver function was assessed by transaminase activity. The metabolic profiles of the hepatocytes were detected using a Seahorse XFe24 extracellular flux analyzer. The direct targets of miR-100 (such as IGF1R-β, mTOR and CDC25A) and HCC related protein (SHP-2) were detected by qRT-PCR and Western blot in liver tissues.
Results: The resultant homozygous knockout mice with genotype of miR-100flox/flox-Alb-Cre+ showed an 80% decrease in hepatic miR-100 expression. In adult mice, miR-100 knockout has no effect on the liver function and morphology. In aged mice, HE staining showed that miR-100 knockout caused infiltration of inflammatory cells and expansion of hepatocellular nuclei. Consistently, liver function was impaired in miR-100 knockout aged mice as indicated by increased serum AST and ALT levels. The metabolic analysis demonstrated that the miR-100 knockout hepatocytes tend to adopt glycolysis. The expressions of the miR-100 target genes, such as IGF1R-β, CDC25A and mTOR, were increased. In addition, the known HCC related protein, SHP-2 also was up-regulated in the knockout livers.
Conclusions: We successfully generated a miR-100 hepatocyte-specific knock-out mouse model. The malignant transformation related to HCC were observed in aged mice. Therefore, this model is suitable for investigating the mechanism of miR-100 inactivation contributing to HCC genesis in vivo.

背景：微小RNA-100（miR-100）的失活参与肝细胞癌（hepatocellular carcinoma, HCC）的发生发展，且miR-100发挥肿瘤抑制因子的功能。为在体内探究miR-100在肝细胞癌发生与进程中的作用机制，我们构建了肝细胞特异性miR-100敲除小鼠模型。
方法：将携带flox位点修饰的miR-100等位基因的纯合小鼠（miR-100flox/flox）与白蛋白启动子驱动Cre重组酶的转基因小鼠（Alb-Cre+/+）交配，获得基因型为miR-100flox/floxAlb-Cre+的纯合子小鼠。分别针对LoxP位点与Cre重组酶设计引物，对小鼠尾部基因组DNA进行基因型鉴定。通过实时定量聚合酶链反应（quantitative Real-time PCR, qRT-PCR）验证肝脏组织中miR-100的特异性敲除效果。采用苏木精-伊红染色（HE-staining）开展组织学分析，通过转氨酶活性检测评估肝功能。使用Seahorse XFe24细胞外流量分析仪（Seahorse XFe24 extracellular flux analyzer）检测肝细胞的代谢谱特征。通过qRT-PCR与蛋白质印迹（Western blot）检测肝脏组织中miR-100的直接靶基因（如IGF1R-β、mTOR及CDC25A）以及肝细胞癌相关蛋白（SHP-2）的表达水平。
结果：最终获得的基因型为miR-100flox/flox-Alb-Cre+的纯合敲除小鼠，其肝脏内miR-100表达量降低80%。在成年小鼠中，miR-100敲除对肝功能与肝脏形态无明显影响。而在老年小鼠中，HE染色结果显示miR-100敲除可引发炎症细胞浸润与肝细胞核扩增；同时血清天冬氨酸转氨酶（AST）与丙氨酸转氨酶（ALT）水平升高，提示老年敲除小鼠肝功能受损。代谢分析表明，miR-100敲除的肝细胞倾向于采用糖酵解代谢模式。miR-100靶基因IGF1R-β、CDC25A及mTOR的表达水平均显著上调，已知的肝细胞癌相关蛋白SHP-2在敲除小鼠肝脏中同样呈现表达上调。
结论：我们成功构建了肝细胞特异性miR-100敲除小鼠模型。老年小鼠体内出现与肝细胞癌相关的恶性转化特征，表明该模型适用于在体内探究miR-100失活介导肝细胞癌发生的分子机制。