The Roles of Tripartite Motif-containing Protein 72 in Oral Cancer Progression

目的:我們的目標在於探討三分膜體包含蛋白72號在口腔鱗狀上皮細胞癌進程中所扮演的角色，並將其與臨床上的資料以及病人的存活率藉由統計方式比較其相關性。除此之外，也針對其詳細的調控機轉作進一步的探究及闡述。 實驗設計:利用SAS、CA922、HSC3、Cal27以及TW2.6五株口腔鱗狀上皮細胞癌細胞株來探討三分膜體包含蛋白72號表現量被小髮夾型核糖核酸抑制或轉殖表現質體過度表現後的移行能力、浸襲能力以及生長能力是否有受到影響。在細胞移動能力的測定上，我們利用博登細胞移行器實驗來做分析;生長能力則是使用细胞存活率分析做測定。動物模式實驗是利用原位種植的技術，使重症聯合免疫缺陷小鼠的頰黏膜生成口腔鱗狀上皮細胞癌腫瘤，並檢驗其淋巴轉移情形和動物存活率。藉由核醣核酸微列陣與GESA分析主要訊息傳遞路徑，並使用pulse-chase assay及泛素化WB-IP分析下游因子與三分膜體包含蛋白72號調控之機制。 結果:臨床上顯示三分膜體包含蛋白72號在晚期腫瘤以及具有淋巴轉移的腫瘤中呈現顯著提升的表現量(P < 0.05)，並且與病人存活率具有統計意義的相關(P < 0.05)。細胞實驗中發現抑制SAS以及CA922細胞中三分膜體包含蛋白72的表現量會使細胞移行能力(P < 0.05)和浸襲能力下降(P < 0.05)但不會改變生長能力。在動物實驗方面，三分膜體包含蛋白72號被抑制的組別其淋巴轉移能力有顯著地下降，而其存活率則呈現顯著地上升 (P < 0.01)。經由核醣核酸微列陣分析以及基因組富集分析找到下游分子為表皮生長因子受體，三分膜體包含蛋白72號被抑制時可觀察到表皮生長因子受體的蛋白表現量明顯下降，但不影響RNA之表現。經由pulse-chase assay以及泛素化WB-IP實驗發現三分膜體包含蛋白72號是藉由後轉譯修飾的方式調控表皮生長因子受體使其不被泛素化降解。藉由過度表現該下游蛋白則可顯著恢復細胞因三分膜體包含蛋白72號表現量被抑制而造成的移動能力下降 (P < 0.05)。雖然在細胞膜修復機制中，三分膜體包含蛋白72號會招募鈣離子，但是在促進癌症進程時卻不與鈣離子有關聯。 結論:本論文確認三分膜體包含蛋白72號可為臨床口腔鱗狀上皮細胞癌病患之惡化預後因子，該基因能增加口腔鱗狀上皮癌之移行、浸襲能力，及實驗動物之淋巴轉移現象。分子機制為藉由後轉譯修飾作用以抑制表皮生長因子受體泛素化降解，以促進癌症進程。Purpose: We aim to examine the roles of tripartite motif-containing protein 72 (TRIM72) in OSCC progression, and to correlate it with patient clinical status and survival rate. Underlying mechanism is also investigated. Experimental design: Oral cancer cell lines, including SAS, CA922, HSC3, Cal27, and TW2.6, were used as in vitro models to investigate cell phenotypes, such as invasion, migration, and proliferation by ectopic expressed-TRIM72 or silenced-TRIM72 (shTRIM72) plasmids transfection. Boyden chamber assay was performed to check migration and invasion abilities. Proliferation ability was identified by MTT assay. Mice were injected with shTRIM72 or vector control SAS cells to examine lymph node metastasis and survival rate. mRNA microarray of stable transfectants elucidated the downstream signaling. EGFR protein pulse-chase assay and ubiquitination WB-IP analysis are used in this study. Results: Clinical data revealed that TRIM72 expression was related to OSCC patient TNM stage, lymph node metastasis, and survival rate (P < 0.05). Transiently transfected SAS and CA922 cells with shTRIM72 plasmids significantly reduced migration and invasion ability (P < 0.05), but did not alter proliferation ability, compared to vector control cells. Furthermore, we established shTRIM72-stable transfectants in SAS cells, and found that silenced-TRIM72 could significantly down-regulate migration and invasion (P < 0.05). In animal study, lymph node metastasis ability was blocked in orthotopic-injected shTRIM72-stable transfectants, and the survival rate was increased compared to vector control clone (P < 0.05). Additionally, we performed high-throughput mRNA microarray analysis and GSEA analysis to identify epidermal growth factor receptor (EGFR) a crucial downstream pathway. TRIM72-enhanced EGFR expression through inhibited ubiquitin-related degradation, which prolonged the half-life of EGFR. Transfected with EGFR expression plasmid could completely restore migration and invasion abilities in shTRIM72 stable transfectants (P < 0.05). Although TRIM72 recruits Ca2+ to repair membrane damage, it enhanced OSCC progression in a Ca2+-independent manner. Conclusions: TRIM72 was a poor prognostic factor in OSCC patents clinically. TRIM72 enhanced invasion and migration ability in OSCC cells, and increased lymph node metastasis in vivo. TRIM72 promoted EGFR expression through inhibited ubiquitin-related degradation in post-translational regulation to cause OSCC progression