肝細胞癌第四號染色體與第十一號染色體長臂異常表現基因之研究：位於染色體4q33之annexin A10、染色體11q23之ApoA-V及11q12之CANP基因

Aberrantly genes expressed at chromosome 4q and 11q in hepatocellular carcinoma: special emphasis on annexin A10 at 4q33, ApoA-V at 11q23 and CANP at 11q12

柳舒祥(Shu-Hsiang Liu)

指導教授 ： 許輝吉

國立臺灣大學/醫學院/病理學研究所/博士(2004年)

https://doi.org/10.6342/NTU.2004.10349

摘要

肝癌是台灣地區常見的惡性腫瘤。在本研究中我們利用mRNA 分示法來篩選在肝癌組織中異常表現的基因，希望能找出肝癌組織中發生變化的重要基因，其中有三個新穎或已發現但尚無論文發表的基因，值得進一步研究，其中有兩個在肝癌組織中經常是低度表現的基因：分別是位於第四號染色體長臂(4q33)的annexin A10 (ANXA10) (後來證實是ANXA10一新穎較短isoform，命名為ANXA10S，GenBank Accession No. AY626137) 與位在第十一號染色體長臂(11q23)的apolipoprotein A-V。另一個則是位於第十一號染色體長臂(11q12.2)，而在肝癌組織中經常是過表現的新穎基因，我們命名為cancer associated nucleoprotein (CANP)。在本研究中，我們以反轉錄聚合

關鍵字

第十一號染色體 ； 第四號染色體 ； 基因表現 ； 肝癌

並列摘要

Hepatocelluar carcinoma (HCC) is the most common fatal malignancy in Taiwan. In an attempt to identify potential genes related to the tumorigenesis and progression of HCC, we used mRNA differential display method to screen the genes aberrantly expressed in HCC. We identified frequent of downregulation of annexin A10 (ANXA10) at 4q33 and apolipoprotein A-V (apoA-V) at 11q23, and overexpression of cancer associated nucleoprotein (CANP), a novel gene of unknown function, located at 11q12.2. This dissertation is to elucidate the clinicopathological significance of the aberrant expressions of these three genes in the progression of HCC using reverse transcriptase polymerase chain reaction (RT-PCR) analyses. Furthermore, we cloned and characterized of the CANP gene. We showed that ANXA10S and apoA-V mRNA were expressed predominantly in adult liver, CANP mRNA was expressed in fetal liver and in various adult and fetal tissues, but the mRNA level was very low in adult liver. The ANXA10S mRNA and apoA-V mRNA levels in HCC decreased in 66% (121/182) and 65.9% (190/288), CANP mRNA was overexpressed in 76% (214/283) of surgically resected unifocal primary HCCs. In addition to their frequent aberrant expression in HCC, the downregulation of ANAX10s and apoA-V was associated with more frequent high AFP levels (P<0.00001 and P=0.001, respectively), grade II-IV tumor (P<0.00001 and P=0.0000, respectively), portal vein invasion (stage IIIB to IV) (P<0.0001 and P=0.000, respectively), and early tumor recurrence (P=0.0007 and P=0.0007, respectively), and hence significantly lower 4-5 year survival rate (P=0.0014 and P=0.001, respectively). Furthermore, ANXA10 and apoA-V downregulation closely correlated with p53 mutation (P=0.024 and P=0.002, respectively), and exhibited synergistic effects with p53 mutation toward more frequent portal vein invasion (P<0.00001 and P=0.000, respectively) and poor prognosis (P=0.0025 and P=0.00004, respectively). These findings suggest that downregulation of ANXA10S and ApoA-V may in cooperation with p53 mutation, and contribute to tumor progression, particularly portal vein invasion of HCC, and unfavorable prognosis. However, the CANP overexpression did not correlate with tumor size, tumor grade and tumor stage, except the early tumor recurrence of HCC after hepatecomy (P=0.041), suggesting that the CANP mRNA overexpression may be an early event during the growth of HCC. Furthermore, we were able to clone the full-length cDNA of CANP. We showed that CANP associated with the nuclear matrix. During the interphase, strong CANP immunoreactivity was detected mainly in the nuclei of the HeLa cells. At pro-metaphase, metaphase and telophase, CANP was seen in the cytoplasm. It came to our surprise that CANP was observed at the midbody as the dividing HeLa cells progressed to late anaphase in cytokinesis. APC/C-mediated ubiquitinylation and degradation require two different activators, Cdc20 and Cdh1. APC/C-Cdh1 is activated during late mitosis and G0/G1 transition . In this study, we showed that CANP protein were degradated by APC/C-Cdh1 complex，suggesting that CANP may participated in late mitosis and G0/G1 transition. We also showed that during cell cycle progression after nocodazole block, CANP mRNA increased significantly upon entry to S phase. By transient transfection for CANP siRNA in HeLa cells, the treated cells exhibited higher proportion of cells at G1 phase as compared with the controls (61.01% vs 47.25%, P=0.005). The CANP siRNA stable transfectants formed relatively smaller colonies in 0.35 % soft agar. These results raises the posiblility that CANP may actively participates in the cell division of HeLa cells.
In this dissertation, we elucidate the clinicopathological significance of the aberrant expressions of these three genes in the progression of HCC, and ANXA10S, apoA-V may provide useful information to clinical doctors while diagnosis, prognosis and making strategies to treat HCC patients. Although the real function of CANP is still unknown, our results suggest that this novel gene may play an important role in the development and/or progression of HCC. Hence, more studies are warranted to better understand the biological functions of of these three genes.

並列關鍵字

chromosome 11q ； hepatocellular carcinoma ； chromosome 4q ； HCC ； apoA-v ； gene expression ； CANP ； annexin A10

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