Yang Dong, Tang Sai, Yang Yan, Yang Fan, Jiang Wengang, Liu Yakun, Zhang Fengyun, Fang Haoshu, Wang Siying, Zhang Yuxia
Department of Pathophysiology, School of Basic Medical Science, Anhui Medical University, Hefei, China.
Front Oncol. 2019 Jun 26;9:535. doi: 10.3389/fonc.2019.00535. eCollection 2019.
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 , we developed hepatocyte-specific miR-100 deficient mice. Mice homozygous for floxed miR-100 allele that carried the Alb-Cre transgene (miR-100Alb -Cre) were developed by mating miR-100 mice with Alb-Cremice. 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. The resultant homozygous knockout mice with genotype of miR-100-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. 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 .
微小RNA-100（miR-100）的失活与肝细胞癌（HCC）有关，且miR-100发挥肿瘤抑制因子的作用。为了解miR-100在HCC发生发展中的功能，我们构建了肝细胞特异性miR-100缺陷小鼠。通过将miR-100小鼠与Alb-Cre小鼠交配，培育出携带Alb-Cre转基因的floxed miR-100等位基因纯合小鼠（miR-100Alb -Cre）。分别使用针对LoxP位点和Cre重组酶的引物对小鼠尾巴DNA进行基因分型。通过定量实时PCR（qRT-PCR）验证肝脏中miR-100的特异性缺失。进行HE染色以进行组织学分析。通过转氨酶活性评估肝功能。使用Seahorse XFe24细胞外通量分析仪检测肝细胞的代谢谱。通过qRT-PCR和蛋白质免疫印迹法检测肝脏组织中miR-100的直接靶标（如IGF1R-β、mTOR和CDC25A）以及与HCC相关的蛋白（SHP-2）。所得基因型为miR-100-Alb-Cre的纯合敲除小鼠肝脏中miR-100表达降低了80%。在成年小鼠中，miR-100敲除对肝功能和形态没有影响。在老年小鼠中，HE染色显示miR-100敲除导致炎性细胞浸润和肝细胞核扩大。一致地，如血清AST和ALT水平升高所示，miR-100敲除的老年小鼠肝功能受损。代谢分析表明，miR-100敲除的肝细胞倾向于采用糖酵解。miR-100靶基因如IGF1R-β、CDC25A和mTOR的表达增加。此外，已知的与HCC相关的蛋白SHP-2在敲除的肝脏中也上调。我们成功构建了miR-100肝细胞特异性敲除小鼠模型。在老年小鼠中观察到了与HCC相关的恶性转化。因此，该模型适用于研究miR-100失活促进HCC发生的机制。
