MiR-193b-3p在人類泌尿上皮癌細胞中CEBPD參與順鉑反應的角色探討

The Role of MiR-193b-3p in CEBPD-Mediated Cisplatin Response in Human Urothelial Carcinoma

林孝任(Siao-Ren Lin)

指導教授 ： 黃阿梅

高雄醫學大學/醫學院/生物化學研究所/碩士(2013年)

https://doi.org/10.6832/KMU.2013.00044

摘要

轉錄因子 CCAAT/enhancer-binding protein delta （CEBPD）最早發現與免疫發炎相關，但也參與在細胞增殖、分化、存活和腫瘤的生成等作用。前人研究指出，CEBPD對於維持基因組穩定和控制乳腺上皮細胞生長扮演重要的角色。順鉑（Cisplatin; CDDP）為以鉑金屬為中心的順式化合物，進入細胞後能與細胞核中DNA鍵結形成加合物而干擾細胞的複製與轉錄作用，為廣泛應用在各種癌症治療的化療藥物，包括人類泌尿上皮癌。然而臨床經驗中，病患在長期接受順鉑治療後，往往會發展嚴重的抗藥性，而導致治療效果不佳。本實驗室先前研究發現順鉑可刺激人類泌尿上皮癌細胞大量表達CEBPD，進而直接轉錄調控銅/鋅-超氧化物歧化酶1 （SOD1）的表現，抑制順鉑所誘導產生的自由基 （ROS）造成抗藥性生成。另外本實驗室前人研究也發現，順鉑可經由誘導 CEBPD進而轉錄調控 PTEN的表現與活性，並使其能於細胞核中累積，且與順鉑產生的細胞毒性有關聯。因此，我們推測 CEBPD在腫瘤發展的進程和順鉑處理產生的反應中可能扮演多重角色；但其詳細機轉仍尚未完全瞭解。微型核醣核酸（MicroRNAs; miRNAs）為非轉譯表現的小片段RNA，由長度約19 ~ 25個核苷酸序列構成，其主要功能為抑制內源性基因之表現。不正常的miRNAs表達已被認為與腫瘤發展有密切關聯性。在本研究中，透過大規模的調查 CEBPD 影響之 miRNAs表達分析，初步結果發現11個miRNA的表達與CEBPD可能有直接相關。其中miR-193b-3p同時可受順鉑刺激而有近兩倍表現之情形。在文獻中提及Cyclin D1和ETS1為 miR-193b-3p所標靶抑制的原癌基因，本研究首次發現透過順鉑誘導所產生的 CEBPD，能夠直接轉錄活化miR-193b-3p，透過了直接與 Cyclin D1和ETS1的 mRNA 3’-UTR序列結合使其表達沉默，並伴隨著細胞移行生長能力被抑制，以及細胞週期停滯在G1時期，同時可敏化泌尿上皮癌NTUB1細胞對於順鉑處理之反應。綜合上述結果，本研究首次揭開 CEBPD透過直接轉錄活化miRNA，而在順鉑反應中扮演影響細胞毒性之角色。本篇研究提供了一個治療尿路上皮癌新的標的與方向參考。

關鍵字

泌尿上皮癌 ； MiR-193b-3p ； CEBPD ； 順鉑

並列摘要

Transcription factor CCAAT/enhancer-binding protein delta （CEBPD）plays multiple roles in tumor progression. Previous studies have demonstrated that cisplatin（CDDP）induced CEBPD expression and led to chemotherapeutic drug resistance. CDDP also activated PTEN expression and nuclear-accumulation and led to CDDP cytotoxicity in human urothelial carcinoma NTUB1 cells. The underlying molecular mechanisms of CDDP-regulated CEBPD expression and its relevant roles in CDDP responses remain elusive. MicroRNAs（miRNAs） are small non-coding RNAs that negatively regulate mRNAs in a sequence-specific manner. The abnormal miRNAs expression is associated with tumor progression. In current study, a large-scale PCR-based miRNA screening was performed to identify CEBPD-associated miRNAs in NTUB1 cells. Eleven miRNAs were selected with more than two-fold changes. MiR-193b-3p, a known tumor suppressor, down-regulated proto-oncogenes Cyclin D1 and ETS1 expression and led to cell cycle arrest, cell invasion and migration inhibition. The result was first confirmed that the miR-193b-3p expression was associated with the DNA binding ability of CEBPD in CDDP response. CEBPD knocking-down approach provided a strong evidence of the positive correlation between CEBPD and miR-193b-3p. CDDP-induced CEBPD trans-activated miR-193b-3p expression and it directly targeted the 3’-UTR of Cyclin D1 and ETS1 mRNA and silenced the protein expression. In addition, miR-193b-3p also inhibited cell migration activity, arrested cell at G1 phase and sensitized NTUB1 to CDDP treatment. In conclusion, this study indicates that CEBPD may exhibit an anti-tumorigenic function through inducing miR-193b-3p expression upon CDDP treatment. This study provides a new direction for managing human urothelial carcinoma.

並列關鍵字

MiR-193b-3p ； CEBPD ； Cisplatin ； bladder cancer

參考文獻

參考文獻

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