摘 要
Wnt/β-catenin signaling pathway在胚胎發展發育及腫瘤的生成過程中都扮演著相當重要的角色。當正常的路徑被不正常的活化時，會導致下游基因不當誘發，並促使腫瘤生成。目前已有多個Wnt/β-catenin target gene 被發現確實與腫瘤的生成有極大的關係，如 c-myc，cyclin D1，MMPs 及survivin 等。因此，了解Wnt/β-catenin target gene 如何調控腫瘤生成是一項重要的議題。實驗室於先前建立了一個結構完整、具擴展性的生物資料庫，簡稱 Bio-database。實驗室將此Bio-database用來尋找有潛力的Wnt/β-catenin target gene。與史丹佛醫學中心所架設的The Wnt homepage 所記錄的109個基因做交集，得到65個Wnt/β-catenin gene，將這些基因設定參數後，從資料庫的初步篩選中得到161個基因，經由文獻的搜尋將其分成111個已知功能基因和50個未知功能基因。而在已知功能基因中發現，確實有高比例的Wnt/β-catenin target gene存在。實驗室先前已挑選25個基因去鑑定是否為Wnt/β-catenin target gene，而接下來要針對另外25個基因去篩選。利用 Lithium chloride 和 Wnt-3A 條件培養液，兩種分別為非特定和特定活化的方式處理 SK-hep-1 細胞的 Wnt/β-catenin 傳遞路徑，再經由半定量 RT-PCR 發現 25 個未知基因中，有5個基因(ETV4、7h3、GPR107、MGC13183及METTL1) 的mRNA在6小時的表現量會增加。接下來進一步探討這5個基因的表現是否能直接被β-catenin/TCF complex所調控，我們利用染色質免疫沈澱法來證實β-catenin/TCF complex會直接結合於ETV4、7h3、GPR107、MGC13183及METTL1的promoter位置。並且對這5個基因進行TCGA database的搜尋，分析肝癌病患的整體存活率(Overall survival)及無病存活率對於肝癌是否有差異。目前結果顯示，利用生物資訊分析結合實驗篩選的方式，可以找到ETV4、7h3、GPR107、MGC13183及METTL1可能為Wnt/β-catenin target gene。
由實驗室先前所篩選的25個基因中發現有3個基因為Wnt/β-catenin target gene，分別為ZNF496、RMI2及C3orf39(出處：陳怡文, 結合生物資訊暨實驗室篩選以尋找Wnt/ β-catenint傳遞路徑之新穎基因, 2011)，其中C3orf39經由Luciferase reporter assay、truncation assay及TCF binding element point mutation後發現C3orf39的β-catenin確實是會被影響的，為了確定C3orf39會影響細胞，我們進行了C3orf39 knockdown實驗，當knockdown C3orf39時，會影響細胞的cell proliferation、migration、invasion、 epithelial-mesenchymal transition及wound healing，確定會影響細胞之function後，接著進行動物實驗，將knockdown C3orf39的細胞注射進入小鼠皮下，結果顯示knockdown C3orf39的小鼠腫瘤體積比對照組小許多。由in vivo的實驗結果中顯示，C3orf39為Wnt/β-catenin target gene。

另外兩個基因ZNF496及RMI2已經由in vitro 的 functional assay證實，當被overexpression時，確實會影響細胞的cell proliferation、migration及invasion…等(出處：張瀞云, Zinc Finger Protein 496在肝癌中是一個有潛力的Wnt/ β-catenint路徑中的標的基因, 2016) (出處：謝旻樺, 尋找及探討具備腫瘤胚特性之新穎生物標記基因並強調於Zinc Finger Protein 496, 2017)(出處：鄭舒文,實驗記錄)。接下來將對這2個基因進行動物實驗，結果顯示在小鼠皮下注射中，經40天以上的觀察發現，被注射overexpression 細胞的小鼠，皮下腫瘤體積與對照組有顯著性的差異。由in vitro及in vivo的實驗結果中顯示，ZNF496及RMI2確實為Wnt/β-catenin target gene。再進一步確認ZNF496是否會有轉移的發生，我們將ZNF496 overexpression的細胞注入小鼠spleen中，觀察是否會經由血液循環轉移進入肝臟及肺臟，結果顯示實驗組的轉移嚴重性與對照組有顯著性的差異，更可以確定ZNF496除了會影響腫瘤本身，更會導致轉移的發生。未來也可以利用IHC染色，對肝癌病患的組織檢體進行ZNF496及RMI2的染色。

總結實驗室目前的基因，從in vitro及in vivo的結果推測C3orf39、ZNF496及RMI2，確實能成為 noval Wnt/β-catenin target genes，在未來實驗中，會利用其三種基因之antibody做IHC染色，觀察在肝癌病患檢體中的結果是否符合預期。除了這三個基因以外，在新篩選的25個基因中，我們實驗室認為MGC13183(CCDC77)是非常具有潛力成為noval Wnt/β-catenin target genes，不僅實驗結果符合預期，且在TCGA中肝癌的整體存活率(Overall survival)及無病存活率(Disease free survival)都有顯著性的差異，對於MGC13183的功能目前屬於未知的狀態，希望MGC13183在未來中能有相當出色的表現。

SUMMARY
Previously, Using bioinformatics to search target gene of Wnt/β-catenin, and setting up the bio-database. The cross comparison of our database and The Wnt homepage (set by Stanford University), Using 65 published genes of Wnt/β-catenin as references to set the thresholds of bioinformatics analysis yielded 161 genes. In the 111 known genes, we indeed found a high percentage of target genes of Wnt/β-catenin. Filtering related conditions, finally we get 50 unknown genes, that having high proportion with Wnt/β-catenin gene. We believe there are target gene of Wnt/β-catenin in the 50 unknown genes. In previously, our lab chose 25 genes to identify Wnt/β-catenin target gene in HCC cell lines with experimental approach. After the identifying, we screen another 25 genes as the same way. In addition to cell functional assay, animal modal is an important method to conform the result. In my studies, we found ETV4, GPR107, MGC13183 and METTL1 ,they have potential to be Wnt/β-catenin target gene. Furthermore, C3orf39, RMI2 and ZNF496 can promote tumourigenesis in vitro and in vivo.
Key words: Wnt/β-catenin pathway, target gene
INTRODUCTION
The Wnt/β-catenin signaling pathway plays an important role in embryogenesis and tumorigenesis. In human cancer, abnormal activity of Wnt/β-catenin signaling pathway induces overexpressed of downstream genes, and initiate oncogene. There are several target genes known to be key players in tumourigenesis, such as c-myc, cyclin D1, MMPs or survivin. Therefore, identifying the target genes of Wnt/β-catenin signaling pathway is important for understanding Wnt/β-catenin-mediated carcinogenesis. Recently, many of researches found Wnt/β-catenin plays an important role in tumorigenesis. In human cancer, abnormal activity of Wnt/β-catenin signaling pathway induces overexpressed of downstream genes, and initiate oncogene. This abnormal pheromone has found in colorectal cancer, liver cancer, ovarian cancer, pancreatic cancer, breast cancer and lung cancer. Hepatocellular carcinoma (HCC) is one of the most malignant tumors and the fourth leading cause of cancer-related death in the world. Our lab developed a combined bioinformatics to find potential target gene of the Wnt/β-catenin signaling pathway. We got the 25 candidate genes via bioinformatics. On the other hand, C3orf39 is the Wnt/β-catenin target gene that we found previously. In this study, we analyze the 25 candidate genes and investigate the function of C3orf39 in human HCC cell line.

MATERIAL AND METHODS
Antibodies for western blot Antibodies used in Western blot include anti- β-catenin(BD Transduction LaboratoriesTM), anti-C3orf39, anti-E-cadherin(Cell signalling), anti-Vimentin(Cell signalling), anti-Twist(Genetex), anti- β-actin(Arigo).
Cell line Human HCC cell line SK-hep-1 and PLC/PRF/5. SK-hep-1 was maintained in DMEM high glucose (HIMEDIA) supplement with 1%AA(Caisson) and 10% FBS(GeneTex), and cultured at 37℃ with 5 %CO2 . PLC/PRF/5 was maintained in MEM (HIMEDIA) supplement with 1%AA(Caisson) and 10% FBS(GeneTex), and cultured at 37℃ with 5 %CO2.
Chromatin immunoprecipitation (ChIP) assay ChIP can be used to identify region of the genome associated with these proteins, or conversely, to identify proteins associated with a particular region of the genome.(Millipore) HCC cell incubate with LiCl at 37℃. After 3 hours, we added 37% formaldehyde to fix the cells, and collected the cells into 1.5 ml eppendorf. Joined cell lysis buffer and nuclear lysis buffer separately after every centrifugation. The eppendorfs were put the corresponding antibody after the sonication of cells. 4℃ overnight, separated the liquid and the protein G beads. Each eppendorfs were washed in 10 times, and added the elution reagent to get genomic DNA.
Cell Proliferation analysis Cell proliferation was measured with a WST-1 solution (Roche). HCC cells infected with lentivirus were seeded in 24-well plates in 500 μl of medium containing 10% FBS and incubated at 37℃ with 5 %CO2. After 24, 48, 72 and 96 hours, the medium was suction with 250 μL of not fresh medium and 25 μL WST-1 solution was added to each well. Cell were then incubated for 4 hours at 37℃ with 5 %CO2, afterward absorbance at 480 nm was measured using a microplate reader. Each experiment was performed in triplicate and repeated in quadruplicate for each condition.

RESULTS AND DISSCUSSION
Wnt/β-catenin signaling pathway is one of the reason that forming liver cancer, we decided using one of the liver cancer cell line, SK-hep-1, to do the experiment. First, we do the cell culture with non-treatment and treatment with Lithium chloride or Wnt-3A conditioned medium, two different methods to activate the Wnt/β-catenin signaling pathway. Collection the RNA and doing Western blot respectively, reversing the RNA to cDNA and done with PCR, which the primers are designed by unknown genes. High expression of the unknown genes become potential of target genes. The mRNA expression of five genes (ETV4, 7h3, GPR107, MGC13183 and METTL1) was found to be up-regulated in cells with activated Wnt/β-catenin pathway. We may do more experiment to confirm the relationship of filtered genes and Wnt/β-catenin signaling pathway. The connection of beta-catenin/TCF complex and unknown target genes, Using the procedures described above, we analyzed five promoters and found the sites within 2.0 kb upstream of the transcriptional start site. Chromatin immunoprecipitation (ChIP) assay was performed to further examine whether the expression of five genes was directly regulated by β-catenin /TCF (T cell factor) complex. The results demonstrated direct interaction of β-catenin/TCF complex with the promoter of four genes (ETV4, GPR107 , MGC13183 and METTL1).

Figure 1 The genes are screening from Lithium chloride and Wnt-3A condition medium.
The genes expression were confirm by RT-PCR.

C3orf39 is a Wnt/β-catenin target gene that we found previously. The gene has expected result in luciferase reporter assay, which conform the ChIP analysis data. When C3orf39 was knockdown, the ability of cell proliferation is decreased and it also downregulate the capability of cancer cell migration and invasion in vitro. In the animal modal, tumor weight in C3orf39 knockdown mice was significant lower than control. These finding confirmed the efficacy of C3orf39 knockdown in suppressing tumor growth in vivo.

Figure 2 C3orf39 is a novel Wnt target gene in vitro and in vivo.
Knockdown C3orf39 decreased migration and invasion in HCC cell line, and also suppressed tumor size.

CONCLUSION
1. We screen those gene’ expression in HCC cell lines, Lithium chloride and Wnt-3A conditioned medium, two different methods to activate the Wnt/β-catenin signaling pathway. Using ChIP to identify four genes as Wnt/β-catenin candidate genes. ETV4, GPR107 , MGC13183 and METTL1 can interact with β-catenin/TCF complex directly.
2. Using western blot and transwell assay to analyze the function and characteristics of C3orf39. The results showed that knockdown C3orf39 could affect cell proliferation, EMT markers, cell migration and invasion. Preclinical animal studies revealed that C3orf39 knockdown cell suppresses tumor growth in vivo.

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