Kakehashi Anna, Chariyakornkul Arpamas, Suzuki Shugo, Khuanphram Napaporn, Tatsumi Kumiko, Yamano Shotaro, Fujioka Masaki, Gi Min, Wongpoomchai Rawiwan, Wanibuchi Hideki
Department of Molecular Pathology, Graduate School of Medicine, Osaka City University, Abeno-ku 1-4-3 Asahi-machi, Osaka 545-8585, Japan.
Department of Biochemistry, Faculty of Medicine, Chiang Mai University, 110 Inthawarorot Rd., Sri Phum, Muang, Chiang Mai 50200, Thailand.
Cancers (Basel). 2021 Mar 10;13(6):1216. doi: 10.3390/cancers13061216.
In the present study, potential molecular biomarkers of NASH hepatocarcinogenesis were investigated using the STAM mice NASH model, characterized by impaired insulin secretion and development of insulin resistance. In this model, 2-days-old C57BL/6N mice were subjected to a single subcutaneous (s.c.) injection of 200 μg streptozotocin (STZ) to induce diabetes mellitus (DM). Four weeks later, mice were administered high-fat diet (HFD) HFD-60 for 14 weeks (STAM group), or fed control diet (STZ group). Eighteen-week-old mice were euthanized to allow macroscopic, microscopic, histopathological, immunohistochemical and proteome analyses. The administration of HFD to STZ-treated mice induced significant fat accumulation and fibrosis development in the liver, which progressed to NASH, and rise of hepatocellular adenomas (HCAs) and carcinomas (HCCs). In 18-week-old animals, a significant increase in the incidence and multiplicity of HCAs and HCCs was found. On the basis of results of proteome analysis of STAM mice HCCs, a novel highly elevated protein in HCCs, cache domain-containing 1 (CACHD1), was chosen as a potential NASH-HCC biomarker candidate. Immunohistochemical assessment demonstrated that STAM mice liver basophilic, eosinophilic and mixed-type altered foci, HCAs and HCCs were strongly positive for CACHD1. The number and area of CACHD1-positive foci, and cell proliferation index in the area of foci in mice of the STAM group were significantly increased compared to that of STZ group. In vitro siRNA knockdown of CACHD1 in human Huh7 and HepG2 liver cancer cell lines resulted in significant inhibition of cell survival and proliferation. Analysis of the proteome of knockdown cells indicated that apoptosis and autophagy processes could be activated. From these results, CACHD1 is an early NASH-associated biomarker of liver preneoplastic and neoplastic lesions, and a potential target protein in DM/NASH-associated hepatocarcinogenesis.
在本研究中，利用STAM小鼠非酒精性脂肪性肝炎（NASH）模型研究了NASH肝癌发生的潜在分子生物标志物，该模型的特征是胰岛素分泌受损和胰岛素抵抗的发展。在这个模型中，对2日龄的C57BL/6N小鼠进行单次皮下注射200μg链脲佐菌素（STZ）以诱导糖尿病（DM）。四周后，给小鼠喂食高脂饮食（HFD）HFD - 60，持续14周（STAM组），或喂食对照饮食（STZ组）。对18周龄的小鼠实施安乐死，以便进行宏观、微观、组织病理学、免疫组织化学和蛋白质组分析。给经STZ处理的小鼠喂食HFD会导致肝脏中显著的脂肪堆积和纤维化发展，进而发展为NASH，并出现肝细胞腺瘤（HCA）和癌（HCC）。在18周龄的动物中，发现HCA和HCC的发生率和多发性显著增加。基于对STAM小鼠HCC的蛋白质组分析结果，选择了一种在HCC中高度上调的新型蛋白质，即含缓存结构域1（CACHD1），作为潜在的NASH - HCC生物标志物候选物。免疫组织化学评估表明，STAM小鼠肝脏的嗜碱性、嗜酸性和混合型改变灶、HCA和HCC对CACHD1呈强阳性。与STZ组相比，STAM组小鼠中CACHD1阳性灶的数量和面积以及灶区域内的细胞增殖指数显著增加。在人Huh7和HepG2肝癌细胞系中对CACHD1进行体外siRNA敲低导致细胞存活和增殖受到显著抑制。对敲低细胞的蛋白质组分析表明，凋亡和自噬过程可能被激活。基于这些结果，CACHD1是肝脏癌前和肿瘤性病变的早期NASH相关生物标志物，也是DM/NASH相关肝癌发生中的潜在靶蛋白。
