肝細胞癌中PANE1基因之臨床病理與功能研究

Abstract

肝癌是台灣地區常見的惡性腫瘤。在本研究中我們從際網路上下載史丹佛大學對165 個樣本肝細胞癌基因微陣列分析所完成的原始數據加以分析，希望能找出在肝癌中發生變化的重要基因，結果篩選出五個在肝細胞癌有過度表現的基因：PANE1 (proliferation associated nuclear element 1) , KIAA0101, PTTG1, LAP18, ZWINT等，並且尚未發現與肝癌相關之正式論文發表，值得作進一步的研究。我們首先以反轉錄-聚合酶連鎖反應 (RT-PCR) 大量篩檢在肝癌組織中這些基因mRNA異常表現的狀況，並與臨床病理學的特徵作關聯性的分析，其中發現PANE1在肝癌組織中過量表現最為明顯，並且在相對非腫瘤肝組織中表現量很低，所以我們以PANE1作為本篇論文的研究重點。目前僅有的兩篇報導顯示PANE1主要表現於免疫細胞或其腫瘤，包括白血病及淋巴瘤並，且在胚胎組織的肝、脾、胸腺組織有相等程度的高度表現，而於成人組織則表現量低。 在臨床病理學的研究上，我們選取125個原發性肝細胞癌作分析，發現到58.4%的原發性肝細胞癌有PANE1的過量表現，並且與多項的臨床病理學特徵都具有顯著的相關性，當PANE1過量表現時，病人血清AFP濃度升高 (AFP>200 ng/mL，71%比 27 %，P值=0.00001)，常出現在較大的腫瘤 (直徑>5 cm， 67%比50 %，P值＝0.054)，當腫瘤級別 (grade) 高 (II,III,IV級) 腫瘤分化差時PANE1表現量高 (97%比44 %，P值＝0.0007)，病人肝癌期別 (stage) 越高且有發生血管侵犯者 (第III、IV期肝癌)，PANE1表現量高 (71%比38 %，P值＝0.0003)。PANE1的過量表現病人容易在一年內早期肝癌復發 (58%，P值＝0.0002) 有關，並且病人的五年存活率 (P值＝0.048) 較低，顯示PANE1與肝癌的分化、侵襲與轉移有密切關連，可以當作肝癌預後因子，且可預測對預後極為不良的早期肝癌復發，有助對術後病人的追蹤與處理。所以我們繼續研究PANE1在癌症發生中扮演的角色及可能具有的功能。 在本研究中，我們用表現載體表現PANE1於細胞株中，以模擬PANE1在肝癌中的過量表現，用MTT assay測定生長速率，發現過量表現PANE1可使細胞在無血清的情況下生長較快；用soft agar assay測定結果發現過量表現PANE1可以促進細胞的非貼附性生長能力；用裸鼠作腫瘤生長能力分析，則過量表現PANE1的細胞株可以在生物體內生成腫瘤；相對地，當我們用PANE1 siRNA使細胞的PANE1 mRNA表現量降低 (knockdown) 時，則使細胞長成群落的能力降低。由這些結果我們可以推測PANE1可能是一個致癌基因。 由於在HeLa細胞週期cDNA微陣列資料庫中，我們發現PANE1 mRNA的表現似乎有隨S期而增加，M期而減少，所以進一步研究PANE1在細胞週期中的變化，我們用EGFP-PANE1表現載體轉染至細胞中，發現PANE1的分佈和細胞週期有關，當細胞在S期時，PANE1主要分佈在細胞核，進入G2/M期時則分佈在細胞質較多，並還有出現在細胞核附近的點狀分佈。而我們也看得到EGFP-PANE1在核膜的分佈，由酵母菌雙雜合系統結果發現到Nup37具有與PANE1的結合潛力並位於核膜可以傳送大分子於細胞核質間，因此推測PANE1經由與Nup37的結合而分佈到不同的位置。另外，我們用西方墨點分析法測定持續表現EGFP-PANE1融合蛋白的細胞株中PANE1-EGFP的量，發現到在細胞週期中的G1/S期與G2/M進入G0/G1期時EGFP-PANE1會被降解，明顯減少，顯示出PANE1的降解與細胞週期有關，而PANE1胺基酸序列上具有D-box並且具有與蛋白酶體26S次單元體ATP酶2及ring-box 1 (RBX1)等與泛素-蛋白酶體途徑相關的結合潛力，而和細胞週期相關的蛋白質亦常由此途徑降解，因此PANE1經由此途徑降解的機會很大。 我們用酵母菌雙雜合系統想要來尋找和PANE1具有交互作用的蛋白質並嘗試瞭解PANE1在生物學上可能的角色。由此結果，我們發現到與PANE1具有結合潛力中的蛋白質中如解旋酶、引子合成酶為DNA合成時需要的酵素及cyclin-dependent kinase 2-interacting protein和細胞週期有關的蛋白質，表示PANE1的確可能細胞週期有關。另一方面，我們也發現到PANE1具有與PLZF轉錄抑制子、SAP30轉錄共抑制子及GTF2IRD2轉錄因子的結合潛力表示PANE1在轉錄調節上具有功能。

Hepatocelluar carcinoma (HCC) is one of the most common fatal malignancies in Taiwan. In an attempt to identify potential genes related to the tumorigenesis and progression of HCC, we downloaded the raw data of cDNA microarray analysis of human HCCs from Stanford cDNA microarray database web site. Based on the bioinformatics analysis, we selected five genes further study, including PANE1 (proliferation associated nuclear element 1), KIAA0101, PTTG1, LAP18, ZWINT, which were shown to aberrantly express in HCC and had not been reported to be associated with HCC. First, we used RT-PCR for large-scale screening of the mRNA expression of these genes in HCC and correlated the aberrant expression with the major clinicopathological features of HCC. We found that PANE1 was overpressed in HCCs, whereas the mRNA level was very low or undetectable in the non-tumor liver. So we chose PANE1 for this study. From the limited information reported in two papers, the expression of human PANE1 gene was detected preferentially in immune cells, including leukemias and lymphomas. PANE1 was highly expressed in fetal liver, thymus and spleen, but little expression was observed in the majority of adult tissues. In the clinicopathologic study of the 125 patients with unifocal primary HCC, the overexpression of PANE1 mRNA was found in 73 (58.4%), and associated with more frequent high serum AFP elevation (71%, P=0.00001), tumor size more than 5 cm (67%, P＝0.054), grade II-IV tumor (67%, P=0.0007), portal vein invasion (stage IIIB to IV) (71%, P=0.00003), and early tumor recurrence (58%, P=0.0002), and hence significantly lower 4-5 year survival rate (p=0.048). These findings suggest that PANE1 overexpression is associated with bigger tumor size, poor tumor differentiation, and greater tumor invasion/metastasis potential, hence more advanced disease stage and poor prognosis. Our results also indicate that PANE1 overexpression can serve as a useful predictive factor for early tumor recurrence, which is one of the most factors associated with poor prognosis of HCC after tumor resection. We furthermore study the role and possible function of PANE1 in tumorigenesis. In this study, we used expression vector to mimic PANE1 overexpression in HCC. Our results indicate that overexpression of PANE1 can enhance cell proliferation on serum free medium by MTT assay, induce anchorage-independent growth by soft agar assay, and induce tumorigenic transformation in vivo by nude mice tumorigenicity assay. This suggestion is supported by the observation that siRNA knockdown of PANE1 reduced colony formation. Hence, it is suggested that PANE1 may be a oncogene. The HeLa cell cycle cDNA microarray’s database indicates that PANE1 is periodically expressed in the HeLa cell cycle. The PANE1 mRNA expression appears to increase in S-phase and decrease in M-phase. We then did a study to analyze the change of PANE1 mRNA level during cell cycle progression. With the transfection of EGFP-PANE1 to Huh7 cell, we found that in S-phase, PANE1 was mostly located in the nuclei, and persisted in the cytoplasm in G2/M-phase, associated with a dot distribution near the nuclei. We also found that PANE1 located to the nuclear envelope. It is possible that PANE1 can be bidirectional transported between the cytoplasm and nucleus. By yeast two-hybrid screen, we identified Nup37 as a PANE1 binding protein. Nup37 is localized to the nuclear envelope (PANE1 shuttles to the nuclei via the binding with Nup37) On the other hand, we found that PANE1 could be degraded in S-phase and late mitosis to G0/G1 transition. PANE1 has a D-box sequence that can bind the ring-box1 protein and proteasome 26S subunit 2, ATPase, which is associated with ubiqutin-proteasome pathway. Cell cycle associated proteins are often degraded through this pathway. It is possible that PANE1 may be also degraded through this pathway. We used yeast two-hybrid to identify the interaction proteins of PANE1 for further studies to better know the biological function of PANE1. We found some that PANE1-interacting proteins, such as primase, helicase and cyclin-dependent kinase 2-interacting protein which play important roles in DNA synthesis or cell cycle progression. We also found that PANE1 interacted with transcription corepressor PLZF and SAP30, and transcription factor GTF2IRD2. These findings suggest that PANE1 play an important role in transcription regulation.