nTAT-DRBD介导“鸡尾酒siRNAs”穿膜的外泌体抗AIPC实验研究

Androgen-independent Prostate Cancer（AIPC） is recognized as a difficulty in the treatment of advanced prostate cancer (PCa). At present, PCa siRNA treatment is confined to a single target, and targeting in the AIPC progress is rarely reported. It may be difficult for AIPC to achieve a good efficacy in finding a single effective gene target. Multiple target interventions are expected to disturb the expression of several key genes in the progress of AIPC, aiming to reversing its phenotype and helping it regaining androgen sensitivity, which help to achieve good tumor inhibition effect combined with anti-androgen therapy. Our previous study focused on improving siRNA delivery efficiency to tumor cells by using transmembrane peptide TAT-DRBD. Combined with the recently discovered capability of exosomes as natural siRNA delivery vector, TAT-DRBD could also be expected to achieve efficient loading of multi-target siRNA cluster into exosomes, with the help of DRBD binding with siRNA in "high affinity, but low sequence dependency ". Therefore, this project aims to use a novel exosomes siRNA loading strategy to explore the possibility of AIPC phenotype reversal of multi-target siRNAs, which will have important clinical significance and application value for AIPC treatment.

雄激素非依赖性前列腺癌（AIPC）被公认是进展期前列腺癌（PCa）治疗的重点和难点。目前PCa的siRNA治疗局限于单一靶点，且靶向AIPC进展过程中的靶点少有报道。对于AIPC，寻找单一有效的基因靶点可能难以取得较好疗效，多靶点干预有望靶向AIPC进展过程中的多个关键基因，逆转其表型，使AIPC细胞重获雄激素敏感性，从而与抗雄治疗联合发挥抑瘤作用。本课题组前期集中于利用穿膜肽TAT-DRBD改进siRNA向肿瘤细胞递送的效率，结合最近发现的外泌体作为siRNA天然递送载体的优势，该策略与siRNA结合“高亲和力，低序列依赖性”的特点也有望实现多靶点siRNA簇向外泌体中的高效加载。所以，本项目旨在运用一种新型的外泌体siRNA加载策略，探索多靶点siRNA逆转AIPC表型的可能性和有效性，对于AIPC治疗有重要的临床意义和应用价值。

目的：细胞外囊泡（EVs）是细胞衍生的膜状纳米颗粒，通过在细胞之间转移生物分子来介导细胞间的通讯。作为天然的运载工具，EVs与现有的治疗性RNA载体相比，可以表现出更高的输送效率，更低的免疫原性和更好的兼容性。它们的治疗用途的一个主要限制是缺乏一种有效和可扩展的方法来为EVs加载感兴趣的治疗性siRNA分子。在这里，我们报告了一种使用聚阳离子膜穿透肽TAT将siRNA封装到EVs中的新策略。方法：在大肠杆菌原核表达系统中将三个TAT短肽与双链RNA结合蛋白DRBD融合表达，获得3TD融合蛋白。 DRBD以不依赖核酸序列的方式与siRNA结合，有助于siRNA靶向多重基因。这在复杂雄激素非依赖性前列腺癌CRPC的多基因靶点（FLOH1，NKX3和DHRS7）治疗中尤其重要。结果：通过超速离心从WPMY-1细胞培养基中分离出EVs。 TIRFM显示改造的EVs（通过3TD加载三种siRNA等比例混合物）中黄色荧光的增加和zeta电位绝对值的降低证明了siRNA与EVs的共定位，这表明TAT可将siRNA成功递送到WPMY-1的EVs中。 qRT-PCR分析显示，相对于未处理的细胞，当用改造EVs处理雄激素抵抗的LNCaP-AI细胞时，FLOH1，NKX3和DHRS7的mRNA水平显着降低。 Western和流式细胞仪检测结果表明，改造EVs可传递siRNA混合物，从而有效下调AR表达并诱导LNCaP-AI细胞凋亡。结论：EVs对siRNA的加载效率和多靶标基因的表达下调表明，在保持递送分子功能的前提下，TAT具有高效的向EVs中加载siRNA的潜力，有望成为一种新型的EVs基因治疗方法。