在正常狀態下，胚胎發育過程中參與的基因在出生後便不再表現，可是當腫瘤生成過程中可能會再度被啟動，這類的基因有人稱它為腫瘤胚 (oncofetal)基因。在臨床應用上，這類的基因是很好的診斷指標，藉由抽血偵測此類的指標基因即可早期發現腫瘤形成，甚至在腫瘤形成前及早預防。而本實驗室基於如此的概念之下，從生物資訊龐大的EST (Expressed Sequence Taq, 表現序列標幟) 資料庫中，找出有可能的腫瘤胚基因，之後藉由RT-PCR的方式篩選出具有腫瘤胚表現型的基因進行研究。而在此篩選中，發現了Lin28B此基因，在文獻裡得知其為一個具潛力的腫瘤幹細胞標記(tumor stem cell marker)。而我們實驗室隨後也去證實它的腫瘤胚幹細胞 (Oncofetal stemness) 性質以及去測試它成為循環腫瘤胚胎幹細胞標記 (Circulation oncofetal tumor-stem–cell-like marker) 的潛在性。最後成功地證實它的確是一個良好的循環腫瘤胚胎幹細胞標記，並且可以利用它將初期 (low-stage)的肝癌 (HCC) 病人分為兩種不同復發情況的族群。而這樣的Marker由於其腫瘤胚的特性：在正常成人組織中不表現或表現量低，相較於一般Marker而言干擾性低，此外又可以將腫瘤細胞區分成兩個次族群：Tumor cell、Tumor stem cell。Tumor-stem-cell被認為具有stem cell self-renew的能力，容易造成轉移的發生。
基於上述，我們結合生物資訊學及一些實驗欲找尋一個具有潛力會在胚胎幹細胞中表現的腫瘤胚基因。而其中一個具有潛力的基因為U41 (Unknown 41)，為本篇論文研究的主題，它是一個已知序列但生物功能未知的基因，PCR篩選結果發現它幾乎在所有被測腫瘤細胞株中表現，但是在正常組織中，僅有某些器官 (例如：胎兒時期的腦、正常的腦、骨髓、男性生殖腺) 表現量較多，其餘器官雖然有表現但明顯減少，而周邊血也幾乎檢測不到。在臨床檢體腫瘤非腫瘤配對檢測結果顯示此基因具有類似腫瘤胚基因的表現型。此外在iPS (Induced pluripotent stem cell) 細胞中也有檢測到U41的表現，總和以上結論我們認定U41極有潛力為一個oncofetal tumor-stem-cell marker，我們將會探討其對於stemness、EMT相關基因的影響。
在本篇研究首先利用RT-PCR來檢測U41四種不同shRNAs的knockdown效力，在結果中的確顯示有knock-down效果，但再利用Real-time PCR 再次檢測發現其實四種不同的shRNA皆沒有knockdown效果，探究其原因為一般傳統PCR只能根據最終PCR濃度來回推模板濃度，無法像real-time PCR一樣及時定量，在每一個cycle中監控放大的濃度。其次在HepG2 Lin28B over-expression cell中和control相比U41的RNA表現量雖然沒有差異性，但是在PLC/PRF/15 EpCAM enrich cell中，U41的RNA表現量比起control組來說上升了約10倍左右，再次證明U41的確和stemness是有相關的。另一方面本篇研究成功clone U41全長，以及完成和EGFP 或是Myc-tag fusion的蛋白，雖然在western blot 中尚未偵測到其蛋白表現，但是在螢光顯微鏡中有觀察到少量的U41-EGFP的螢光表現，推測是片段太大不容易以短暫感染使其表現。
目前對於U41的功能始終尚未釐清，有待後續繼續研究解謎。

Our laboratory devotes to the study of unknown oncofetal genes (genes expressed in embryos or fetuses, down-regulated or undetectable in adult tissues, and re-expressed in tumors). We used a bioinformatics algorithm to screen for potential oncofetal genes using the expressed sequence tag (EST) database. One of these genes, Lin28 homolog B (LIN28B), had been reported to be a potential marker of tumor stem cells. We sought to verify the oncofetal-stemness characters of LIN28B and test its potential as a circulating tumor-stem-cell-like marker in adult HCC patients. The results showed that, by testing LIN28B expressed in circulating cells, we could further stratify low-stage HCC patients into two groups with significantly different recurrence curves.
The results of LIN28B demonstrated the potential significances of oncofetal tumor-stem-cell markers. This kind of marker is low background and can divide tumor cell into two groups: tumor cell and tumor stem cell. Tumor stem cell is thought to cause metastasis by the ability of self-renewal. Therefore we searched for potential oncofetal genes that are also expressed in embryonic stem cells through a combined bioinformatics and experimental approach. One of the candidate genes is U41. Our experimental results revealed that U41 is present in high volumes in newborn and embryos of rats, but no or lower volumes in mature ones. It is overexpressed in lung and liver cancers, but not detected in peripheral blood cells, making it a potentially ideal oncofetal marker.
This study aims to validate U41 as a novel oncofetal tumor-stem-cell marker. We will investigate the functions and characteristics of U41 by over-expression or knock-down experiment using human cell lines. Assays regarding to oncogenesis and stem cell characteristics will be performed. In the knock-down part; the stable pools with U41 knock-down in a human liver cancer cell line (HepG2) and a lung cancer cell line (A549) were established. The mRNA levels of U41 in stable knock-down pools of HepG2 and A549 cell lines were tested by RT-PCR. In the result it seems that the shRNAs are workable. However, we use quantitative real-time PCR to validate it again; we found the shRNAs are not as useful as in knock-down U41.
In the overexpression part; the full-length of U41 in pSP72 vector was constructed successfully. Since the commercially available antibodies of U41 do not work for Western blotting, we subsequently fused full-length U41 with the EGFP gene or a Myc tag in pMSCV (the retrovirus vector). In fluorescence microscopy, U41-EGFP showed some expression, indicating that the U41-EGFP construct should be intact but its expression was low. Although we still cannot detect the U41 which fused with Myc or EGFP in protein level, we would make retrovirus to transfect cell that provide more efficiency than transient transfection.
In summary, the function of U41 is still unknown which needs further study.

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