多功能銀奈米團簇載體應用於乳癌細胞之抑癌微小RNA藥物傳遞與影像追蹤

Abstract

儘管醫療技術在過去幾十年間有很大的躍進，乳癌仍然是全世界婦女主要的死亡原因之一。因此迫切需要發展新穎的癌症療法以挽救更多的生命。微小RNA (microRNAs) 由於可抑制腫瘤基因，已被認為是下一代腫瘤治療法。藉由取代或導入具抑癌特性的microRNA模擬藥物可使腫瘤得到控制，並可達到降低毒性、提高效率、減少脫靶效應的發生。 在本研究中，我們設計了具多功能性的寡核苷酸骨架，合成以銀奈米團簇 (AgNC) 為追蹤探針之藥物載體。所設計的寡核苷酸序列包含一可專一辨識mucin 1 (MUC1為一在MCF-7乳癌細胞膜上高度表現之醣蛋白)之適體序列，一段重覆的胞嘧啶序列，用來合成具有螢光的AgNC，以及一段用於乘載治療基因的互補序列。過去研究已知，MicroRNA-34a (miR-34a)為一抑癌基因，在許多癌細胞內，其表達的量相當低或根本沒有。因此，可藉由重新導入此一基因作為治療藥物分子，抑制致癌基因的表達，並誘導腫瘤細胞凋亡。由共軛焦螢光影像顯示，追蹤AgNC自身的螢光證明建構之奈米載體(MUC1-AgNCm-miR-34a)能有效地進入MCF-7。進一步評估此奈米載體是否能有效傳遞microRNA並具生物活性，我們也對miR-34a下游的目標基因（Bcl-2，Cdk6和Ccnd1）表現和其蛋白質表現分別以定量聚合酶鏈鎖反應和西方墨點進行分析，確認其能有效地抑制癌細胞生長。我們預期此新穎多功能的AgNC奈米載體，對於未來改善乳癌治療診斷將深具潛力。

Despite huge progress in medical technologies in the past decades, breast cancer remains one of the leading causes of death in women worldwide. Novel therapeutics is of urgent need to save more lives. MicroRNAs acting as tumor suppressors have been recognized as next generation tumor therapeutics since the restoration of tumor suppressive microRNAs using microRNA replacement or mimics may be a less toxic, more effective strategy due to fewer off-target effects. In this study, we designed the oligonucleotide scaffold with multifunctionality to synthesize a novel silver nanocluster (AgNC)-based drug carrier. The designed oligonucleotide is composed of a tumor targeting aptamer sequence specific to mucin 1 (MUC1, a highly expressed membrane protein in MCF-7 mammary carcinoma), a poly-cytosine region for fluorescent AgNC synthesis and a sequence for gene loading. MicroRNA-34a (miR-34a), which had been known to be down regulated or lost in various carcinomas, was employed as the therapeutic molecule to inhibit oncogene expression and induce the apoptosis of carcinoma. Confocal images demonstrated constructed nanocarrier (MUC1-AgNCm-miR-34a) could efficiently enter MCF-7 by monitoring the intrinsic fluorescence of AgNC. To evaluate the efficacy of such nanocarrier for microRNA delivery, we investigated the downstream expression of miR-34a targets (Bcl-2, Cdk6, and Ccnd1) in gene and protein levels respectively by quantitative PCR and western blot, validating the effective inhibition by miR-34a. We foresee the potential of this novel multifunctional AgNC-based nanocarrier to improve the breast cancer theranostics.