以誘餌寡核苷酸抑制MUC1-C / KLF4複合體 與誘導人類乳癌細胞凋亡之研究

Abstract

上皮黏蛋白1-羧基端（MUC1-COOH; MUC1-C）與Kruppel-like factor 4 (KLF4) 轉錄因子皆與癌細胞凋亡具有高度的相關性。PE21區域位於腫瘤抑制基因p53之啟動子（promoter）中；而MUC1-C 會與KLF4形成二聚體（MUC1-C/KLF4）鍵結至PE21區域進而抑制p53表現。本研究設計之誘導寡核苷酸（decoy oligonucleotide; dODNs）為雙股具有21個核苷酸，其序列由PE21區域演繹而得。本研究探討dODNs-5’與dODNs-3’兩種序列，鍵結在MUC1-C/KLF4複合體，去阻斷MUC1-C/KLF4複合體與PE21之間鍵結作用，進而活化p53基因，導致人類乳癌細胞（MCF-7）凋亡。在細胞凋亡實驗中，將兩組dODNs轉染至MCF-7 中分別使29.4%與24.5%的細胞凋亡。同時研究將 dODNs的序列隨機組合之新序列為負控制組（scr-1與scr-2），對照實驗顯示生存率依序為83.3%、81.5%。其中空白實驗組（blank）的生存率為81.1%。此外，在基因表現方面，可分別促進p53基因表現達2.99與2.72倍。進一步針對dODNs進行毒性測試，將dODNs轉染至人類纖維母細胞（fibroblast）生存率皆在90%以上。最後，我們也利用免疫染色法，顯示出兩組dODNs位於細胞核內，說明了本研究在驗證機制上，dODNs可進入細胞核抑制MUC1-C/KLF4複合體與PE21作用。由以上結果顯示，本研究提出一個策略以MUC1-C/KLF4複合體為標靶的dODNs，具有選擇性地使MCF-7凋亡。

The MUC1 COOH-terminal subunit (MUC1-C) and the Kruppel-like factor 4 (KLF4) transcription factor are both highly associated with cell apoptosis. The MUC1-C binds to KLF4 to form the MUC1-C/KLF4 complex, which represses p53 transcription by binding to the PE21 element within the tumor suppressor p53 promoter. In this work, we designed two 21-mer double-stranded decoy oligonucleotides (dODNs-5’ and dODNs-3’) based on the MUC1-C/KLF4 PE21 element. Blocking the interaction between MUC1-C/KLF4 and PE21 by the dODNss resulted in up-regulating p53 gene expression and inducing human breast cancer cell apoptosis. In apoptosis assay, transfection of the dODNs-5’ and the dODNs-3’ respectively resulted in 29.4% and 24.5% apoptosis. The viability of transfection of the negative control scrambled-sequence oligonucleotides (1-scr and 2-scr) and the blank control are 83.3%、81.5% and 81.1% sequentially. Both of the dODNs-5’ and dODNs-3’ up-regulated p53 gene expression 2.99 and 2.72 times, respectively. Besides, we chose the fibroblast cells served as a normal cell control. In the fibroblast cells, transfection of the dODNss and the SCRs showed that the viability were all at least 90% in the apoptosis assay. We also showed that the dODNss interacted with MUC1-C/KLF4 in nucleus by immunofluorescence assay. In comparison, the SCRs localize at the outside of the nucleus. To conclude, inhibition of the MUC1-C / KLF4 complex by using a transcription factor decoy can induce human breast cancer cell apoptosis.