AKR1C3誘導口腔鱗狀上皮細胞癌上皮-間質轉換與幹細胞特性之研究

Abstract

依據我國 107 年衛福部統計結果，口腔癌高居國人男性十大癌症中死亡率第 四位。口腔癌的發生主要與吸煙、喝酒和嚼食檳榔有關。檳榔含有的檳榔鹼 (Arecoline)是引起口腔癌的主要致病因子。實驗室先前研究發現，口腔癌病人檢體 中 Aldo-keto-reductase family 1，member C3 (AKR1C3)的蛋白表現量顯著增加，並 且與腫瘤大小和癌症分期呈正相關，患者的五年存活率普遍較低。但其與口腔癌癌 化機轉的關係目前尚未清楚。本研究先將 AKR1C3 的質體與空載體分別送入內生 性 AKR1C3 蛋白表現量低的 Ca922 細胞。根據 transwell migration assay 結果顯示， AKR1C3 表現量高的細胞之移動能力較強。藉由 sphere forming assay 發現過度表 現 AKR1C3 可增加 Ca922 細胞形成聚球體 (sphere) 的能力。此外，AKR1C3 表現 量增加會使上皮-間質轉換 (EMT) markers 中的 N-cadherin 表現量提升，並使 EMT markers 中的 ZO1 和 E-cadherin 表現量降低。同時，AKR1C3 過表現會上調癌幹細 胞相關標誌中 CD133、CD44、KLF4、SOX2 和 Nanog 之表現。以 siRNA 將 AKR1C3 表現量較多的 SAS sphere 進行 AKR1C3 knockdown 後發現，SAS spheres 的形成數 量明顯降低，EMT 中 ZO1 表現上升且同時下調 Snail 和 Twist，而分析其癌幹細胞 相關標誌後發現，CD133、CD44 及 nanog 的蛋白表現皆下降。抗藥性實驗顯示， AKR1C3 表現量高的細胞在加入化療藥物 5-FU 和 cisplatin 作用 72 小時後，分別 於濃度 0.625、1.25 μg/ μl 與 1.25、2.5 μM/ ml 下較對照組都有顯著抗藥效果。綜合 以上結果可知 AKR1C3 表現量高會使細胞發生 EMT 並上調癌幹細胞相關標誌， 進而促進癌細胞的形成細胞 spheres 之能力、生長和移動速度並對 5-FU 和 cisplatin 產生抗性。

According to the statistics of the Ministry of Health and Welfare from Taiwan in 2018, oral cancer is the fifth leading cause of cancer-related deaths. The rate of all the cancer-related deaths of oral cancer rose from 9.6% in 2008 to 12.8% in 2018. The main risk factors for the development of oral cancer are including alcohol consumption, smoking and areca nut (AN) chewing. Among these, arecoline which is the main areca nut alkaloid is one of the carcinogens of oral cancer. In previous study, we found Aldo- keto reductase 1 member C3 (AKR1C3) was highly expressed in oral cancer and was related to the tumor sizes, cancer stages and patients’ 5 years survival rates. However, the mechanisms are still not clear. We first transfected AKR1C3 cDNA plasmid and vector into Ca922 cells to establish stable AKR1C3 overexpression clones. We found AKR1C3 overexpression cells has stronger ability of migration through transwell migration assay, increases sphere forming ability, the expressions of stemness marker proteins such as CD133, CD44, KLF4, SOX2 and Nanog. We also found AKR1C3 overexpression increased N-cadherin expression and decreased ZO-1, E-cadherin protein expression. Otherwise, we found knockdown AKR1C3 in SAS sphere by siRNA could decrease the ability of sphere forming, upregulate ZO1 and inhibit the expression of Snail and Twist, down regulate the expression of stemness markers, such as CD133, CD44 and nanog. In addition, we found AKR1C3 overexpression increased resistance to cisplatin and 5-FU compared to controls. In conclusion, our results indicate that overexpression of AKR1C3 make oral cancer cells to be more migrative, growth faster, easier to form spheres, resistant to cisplatin and 5-FU through enhances stemness and EMT.