Wnt/β-catenin傳遞路徑是一條具高度保留特性的多效性路徑，在胚胎發育（embryonic development）、成熟個體組織恆定（tissue homeostasis）和腫瘤形成（tumorigenesis）中扮演著非常重要的角色。Wnt/β-catenin 傳遞路徑的異常活化會導致下游基因過度表達，如c-myc和cyclin D1，而這些基因通常與許多癌症相關，包括肝癌（hepatocellular carcinoma，HCC）、大腸癌（colorectal cancer，CRC）和子宮內膜癌（endometrial cancer，EC）等。肝癌是造成全球癌症死亡的第四大常見原因，有許多研究證據顯示，Wnt/β-catenin 傳遞路徑的異常活化在肝癌的發生（cancer initiation）和發展（cancer progression）中扮演著重要的角色。因此，確定Wnt/β-catenin傳遞路徑標靶基因對於Wnt/β-catenin傳遞路徑介導的癌症形成（carcinogenesis）是非常重要的。先前，本實驗室建立了一個生物資料庫，簡稱Bio-database，與史丹佛醫學中心建立的The Wnt homepage進行比對後，獲得總共50個可能與Wnt/β-catenin傳遞路徑相關的未知功能基因。經過氯化鋰（Lithium chloride）條件培養基、Wnt-3A 條件培養基和染色質免疫沉澱（chromatin immunoprecipitation，ChIP）篩選，並利用冷光素酶報告基因檢測（luciferase reporter assay）、截斷實驗（truncation assay）和點突變實驗（point mutation assay）驗證後，確定了三個新穎Wnt/β-catenin傳遞路徑標靶基因，其中一個為POMGNT2基因，其蛋白Protein O-linked-mannose beta-1,4-N-acetylglucosaminyltransferase 2參與了α-肌營養不良醣蛋白（α-dystroglycan）中O-甘露醣三醣（O-mannosyl trisaccharide）的生合成，對於結合含有層連結蛋白G樣結構域（laminin G-like domain）的細胞外基質（extracellular matrix，ECM）蛋白扮演著重要的角色。另外，實驗結果已證實減弱（knockdown）POMGNT2會降低人類肝癌細胞PLC/PRF/5的細胞增生（proliferation）、遷移（migration）、侵襲（invasion）和傷口癒合（wound healing）。在動物實驗中，減弱POMGNT2也會抑制腫瘤大小。在本研究中，發現過表達（overexpression）POMGNT2會促進人類肝癌細胞Hep3B和HepG2的細胞增生、遷移和傷口癒合。而與減弱POMGNT2結果一致，過表達POMGNT2在上皮間質轉化（epithelial-mesenchymal transition，EMT）中與對照組相比沒有顯著差異。為了探討POMGNT2在O-醣基化（O-glycosylation）所扮演的角色，利用層連結蛋白覆蓋實驗（laminin overlay assay）搭配西方墨點法（western blotting）進行分析，結果顯示與對照組相比，過表達POMGNT2肝癌細胞中laminin和-DG的蛋白表現量並無顯著差異。綜合以上實驗結果，Wnt/β-catenin傳遞路徑新穎標靶基因POMGNT2確實會影響肝癌細胞的增生和遷移能力。

Previously, our laboratory developed a biological database and compared with the Wnt homepage set up by the Stanford Medical Center. Taking these data together, we identified a total of 50 unknown genes that are likely to be associated with the Wnt/β-catenin signaling pathway. After screening by the Lithium chloride, Wnt-3A conditioned medium and ChIP assays, and validated by luciferase reporter assays, truncation assays, and point mutation assays, we identified three novel Wnt/β-catenin signaling pathway target genes such as Protein O-linked-mannose beta-1,4-N-acetylglucosaminyltransferase 2 encoded by POMGNT2. It is involved in the biosynthesis of the carbohydrate structure phosphorylated O-mannosyl trisaccharide present in α-dystroglycan (α-DG), which is essential for binding laminin G-like domain-containing extracellular proteins. In addition, we confirmed that POMGNT2 shRNA decreased the proliferation, migration, invasion and wound healing of PLC/PRF/5 hepatocellular carcinoma cell lines. In an animal model, POMGNT2 knockdown also suppressed the tumor sizes. In this study, we confirmed that POMGNT2 overexpression enhanced the proliferation, migration and wound healing of Hep3B and HepG2 hepatocellular carcinoma cell lines. Consistently, knockdown and overexpression of POMGNT2 are not significantly different in epithelial-mesenchymal transition (EMT). To identify the role of POMGNT2 in O-linked glycosylation, immunoblot analysis and laminin overlay of WGA-enriched glycoproteins revealed that the expression level of laminin and α-DG are not significantly different between control and POMGNT2 overexpression cells. In summary, the novel Wnt/β-catenin signaling pathway target gene POMGNT2 is related to cell proliferation and migration of hepatocellular carcinoma cells.

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