MicroRNA-17-5p Regulation of Apoptosis-Related Protein Expressions in Oral Squam ous Cell Ca rcinoma Cells Is Related to Betel Quid Chewing

微核醣核酸17-5p調控嚼食檳榔引起的口腔鱗狀細胞癌的凋亡蛋白表現

林其嶪(Chi-Yeh Lin) ； 陳俊佑(Chun-You Chen) ； 陳秋萍(Chiu-Ping Chen) ； 周志成(Chih-Cheng Chou) ； 陳梓欣(Tzu-Hsin Chen) ； 陳薏茹(Yi-Ju Chen) ； 王豪(Hao Wang) ； 李俊毅(Chun-I Li) ； 蔡佳宜(Chia-Yi Tsai) ； 陳加惠(Chia-Hui Chen) ； 吳思遠(Szu-Yuan Wu)

《放射治療與腫瘤學》 22卷4期 (2015/12) Pp. 255-266

https://doi.org/10.6316/TRO/201522(4)255

摘要

前言：在台灣口腔癌與嚼食檳榔是有相關的，放射治療是其中重要的一種治療方式。過去的研究證實微核醣核酸17-5p（microRNAs 17-5p；miR 17-5p，一種RNA的分子）在經由放射線照射過後的鱗狀癌細胞株OC3（長期嚼食檳榔但不抽菸之口腔鱗狀細胞癌之細胞株）之中會表現出來，還發現miR 17-5p會抑制下游蛋白p21的表現並增加放射線的敏感度。本篇研究中，我們利用了數組人類凋亡蛋白釐清了相關蛋白對於微核醣核酸17-5p的調控。材料和方法：細胞溶解產物均來自於具有小干擾RNA或微核醣核酸17-5p鱗狀癌細胞株OC3，並分析其凋亡蛋白的表現。微核醣核酸17-5p相關之凋亡蛋白會再利用西方墨點法Westernblotting來確認。利用放射線照射無p53和有高度的p53鱗狀癌細胞株OC3，再接受碘化與流式細胞儀的檢驗。結果：使用人類凋亡蛋白組（R&D Systems; catalog # ARY009），我們觀察到在放射線照射後的鱗狀癌細胞株OC3中，在微核醣核酸17-5p相對反義寡核甘酸或控制組寡核甘酸的調控鱗狀癌細胞株OC3下有35種凋亡蛋白具有其相對應的反應。其中一些蛋白質，包括p21, p53, TNF RI,FADD, cIAP-1, HIF-1α,與TRAIL R1，這些模組的增強與減弱表現方式與使用西方墨點法確認後是相同的。於此我們更進一步釐清p53在鱗狀癌細胞株OC3經放射線照射過後的角色，我們將p53過度表現，證實鱗狀癌細胞株OC3中p53的表現增加會阻斷癌細胞進入G2/M周期的效果是顯著的。結論：在鱗狀癌細胞OC3中，凋亡蛋白p21、p53、TNF RI、FADD、cIAP-1、HIF-1α和TRAIL R1表現是會受到微核醣核酸 17-5p 調控的。對於鱗狀癌細胞株OC3中，調控促進p53蛋白表現，對於因嚼食檳榔引起的口腔癌細胞放射線敏感度的調節是有影響的。

關鍵字

微核醣核酸17-5p ； 放射線 ； OC3細胞 ； p53蛋白

並列摘要

Background and purpose : Betel nut chewing is associated with oral cavity cancer in Taiwan. Radiotherapy is one of the therapeutic approaches. Our previous study demonstrated that microRNA (miR)-17-5p (one of the miR-17-92 polycistronic miRNAs) was enhanced in the irradiated OC3 cancer cell line (which was established from oral squamous cell carcinoma in a long-term betel nut chewer who did not smoke), and miR-17-5p was also found to inhibit downstream protein p21 expression and induce radiosensitivity. In this study, we used a human apoptosis protein array to clarify which apoptosis-related proteins are modulated by miR-17-5p. Materials and Methods : Total cell lysates from OC3 cells with control small interfering (si)RNA or miR-17-5p were used to analyze apoptosis-related proteins. A specific miR- 17-5p effector apoptosis-related protein was confirmed by Western blotting. To confirm the role of p53 in irradiated OC-3 cells, OC3 cells without or with a p53-overexpressing clone were irradiated and examined by propidium iodide staining and flow cytometry. Results : Using a human apoptosis protein array analysis (R&D Systems; catalog # ARY009), we simultaneously detected the relative expressions of 35 apoptosis-related proteins in OC3 cells that were treated with an miR-17-5p antisense oligonucleotide (AS-ODN) or a control ODN. Several proteins, including p21, p53, TNF RI, FADD, cIAP-1, HIF-1α, and TRAIL R1, were found to be up- or downregulated by miR-17-5p in OC3 cells; their expression patterns were also confirmed by Western blotting. We further clarified the role of p53 in irradiated OC3 cells, using a p53 overexpression strategy, and results revealed that enhancement of p53 expression significantly enhanced radiation-induced G2/M arrest of OC3 cells. Conclusions : miR-17-5p regulated the apoptosis-related proteins of p21, p53, TNF RI, FADD, cIAP-1, HIF-1α, and TRAIL R1 in OC3 cells; interestingly, its effect on p53 protein expression contributed to modulating the cell cycle arrest of OC3 cells.

並列關鍵字

MicroRNA (miR)-17-5p ； Radiation ； Oral carcinoma 3 (OC3) cells ； Tumor protein p53