Analysis of how liver-specific transcriptional regulation factors are implicated in hepatocytic differentiation and oncogenesis ; their application for histo- and clinicopathological diagnosis

分析肝脏特异性转录调控因子如何参与肝细胞分化和肿瘤发生；

基本信息

批准号：
07457050
负责人：
HAYASHI Yoshitake
金额：
$ 5.06万
依托单位：
KOBE UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

项目摘要

Hepatic nuclear factor 1 (HNF-1), as a transcription regulator, binds the promoters or enhancers of genes expressed almost exclusively in liver. We have previously reported that the ratio of HNF-1alpha and HNF-1beta mRNA is related to histological differentiation of hepatocellular carcinoma (HCC) (Ninomiya T et al., J Hepatol, 25 : 445-453,1996). Furthermore, to investigate the expression pattern in HCCs, we relatively quantitatively compared the expression of HNF-1alpha and HNF-1beta proteins in various histologically differentiated cancerous as well as surrounding noncancerous tissues and their binding activity for B element in alpha-fetoprotein enhaner. The polyclonal antibodies for human HNF-1alpha and HNF-1beta were generated by Glutathione S-transferase fussion protein. Nuclear extracts were prepared from the tested tissues. The expression of HNF-1alpha and HNF-1beta proteins in isolated nuclei were measured by western blotting and their binding activity examined by gel mobility assay. Immunohistochemistry were performed in frozen and parafin embedded sections. Western blottings demonstrated that HNF-1alpha protein was overexpressed in well differentiated HCC tissues but decreased from well differentiated to poorly differentiated HCCs compared with surrounding non-HCC tissues. The HNF-1beta expression did not after from well differentiated to poorly differentiated HCCs athough it is more abundant in cancerous tissues than the surrounding noncancerous portions. In gel mobility assay, the decreasing tendency was shown in the assay of HNF-1alpha binding activity. These findings provide an evidence involved in effect of HNF-1alpha alteration on the differentiations of HCC that the histological differentiation of HCC turns poor when HNF-1alpha expression decreases and HNF-1beta competes to bind the elements formerly occupied by HNF-1alpha (Wang W et al., J Pathol in press).

肝核因子 1 (HNF-1) 作为转录调节因子，与几乎只在肝脏中表达的基因的启动子或增强子结合。我们先前报道过HNF-1α和HNF-1βmRNA的比率与肝细胞癌(HCC)的组织学分化有关(Ninomiya T等人，J Hepatol，25：445-453，1996)。此外，为了研究HCC中的表达模式，我们相对定量地比较了各种组织学分化的癌组织以及周围非癌组织中HNF-1α和HNF-1β蛋白的表达及其与甲胎蛋白增强剂中B元件的结合活性。人 HNF-1α 和 HNF-1β 的多克隆抗体是由谷胱甘肽 S-转移酶融合蛋白产生的。从测试的组织中制备核提取物。通过蛋白质印迹法测量分离的细胞核中 HNF-1α 和 HNF-1β 蛋白的表达，并通过凝胶迁移率测定检测它们的结合活性。免疫组织化学在冷冻和石蜡包埋切片中进行。 Western印迹表明，HNF-1α蛋白在高分化HCC组织中过表达，但与周围非HCC组织相比，从高分化到低分化HCC下降。尽管HNF-1β在癌组织中比周围的非癌部分更丰富，但从高分化HCC到低分化HCC，HNF-1β的表达并不明显。凝胶迁移率测定中，HNF-1α结合活性测定呈下降趋势。这些发现提供了HNF-1α改变对HCC分化影响的证据，即当HNF-1α表达减少且HNF-1β竞争结合先前被HNF-1α占据的元件时，HCC的组织学分化变差（Wang W等人，J Pathol 正在出版）。