黑色素瘤威罗菲尼耐药细胞的核酸适体筛选、靶标鉴定及其抗耐药性研究

Current treatment of metastatic melanoma that uses mutant BRAFV600E-specific inhibitor-Vemurafenib (Plx4032) has produced high response rates but with rapid relapse; the addition of a MEK1/2 inhibitor has led to a modest increase in survival. Nevertheless, the majority of tumors progress in most patients. The acquired resistance involves NRAS overexpression-mediated reactivation of MAPK pathway. However, the underlying mechanisms responsible for up-regulation of NRAS in acquired resistance are not clear. Decades of work have not yet yielded any efficient way to block the expression and activity of NRAS. Therefore, identification of novel resistance associated-target genes and therapeutic option are urgently needed.. In order to obtain the specific identification of melanoma Plx4032-resistant cell aptamer, we synthesized ssDNA library and conducted the cell-SELEX screening with the Mel28-Plx4032 resistant cell line (Mel28-PLX) as target cells, and with Mel28 cell line as control cells. After 15 rounds of screening, we found that the LL4 was able to specifically bind to the resistant Mel28-PLX, with no binding to the parental Mel28. By reducing the end of sequences gradually, we analyzed and optimized the sequenced aptamer LL4 (LL4a). Flow cytometry showed that the LL4a exhibited stronger binding capacity with target cell Mel28-PLX. To investigate the possible application of LL4a in complex biological environment, we used a mouse xenografted tumor model. LL4a signal was beautifully enriched in tumor location 5 minutes after tail vein injection and lasted until 120 minutes after injection. Only the mel28-PLX tumors attracted LL4a, and the mel28 showed no signal. Therefore, the aptamer in complex environment can still keep good recognition ability, and maintain a high level of tissue specificity. . At present, there are few cell surface biomarkers for melanoma Plx4032-resistant cells. It is necessary to find a new prognosis biomarker for Plx4032 treatment. In this project, using aptamer-pull down, SDS-PAGE and mass spectrometry, Astrocyte elevated gene-1 (AEG-1), also known as metadherin (MTDH) was selected as a potential target protein of LL4a. As a multifunctional mediator of carcinogenesis, MTDH/AEG-1 has been found to be involved in multiple signaling pathways, such as PI3K/Akt, NF-κB, Wnt/β-catenin and RAS/MAPK. Overexpression of MTDH is observed in a variety of cancers, and has crucial relevance with cancer progression, including initiation, proliferation, invasion, metastasis and chemoresistance, but the function of MTDH is not well studied in melanoma. Our preliminary results show that the membrane protein level of MTDH is significantly increased in Mel28-PLX, indicating MTDH is involved in the pathogenesis of Plx4032-resistance. . Therefore, we hypothesize that LL4a binds to melanoma-plx4032 resistant cells via interaction with plasma membrane protein MTDH, which is able to enhance Plx4032 resistance through activation of NRAS, leading to reactivation of MAPK pathway. Constructing the LL4A-LPD (Liposome-protamine-calf thymus DNA)-MTDH/N-RAS siRNA nanoparticle is a new therapeutic strategy for overcoming Plx4032-resistance in melanoma.

NRAS/MAPK通路重激活是导致黑色素瘤威罗菲尼（Plx4032）耐药的主因，但与耐药相关的细胞膜表面标志物却未见报道。前期基于Cell-SELEX技术，以人黑色素瘤 Plx4032耐药株Mel28-PLX为靶标，我们获得了能特异结合耐药株的ssDNA适体LL4a，通过适体-pull down与LC-MS/MS技术筛选出与RAS/MAPK通路活性密切相关的MTDH（metadherin）蛋白, 且MTDH在Mel28-PLX细胞膜上显著高表达。因此本课题提出科学假说：LL4a特异结合Plx4032 耐药株膜蛋白MTDH，MTDH可增强MAPK信号通路及激酶NRAS的活性，导致耐药产生。本项目拟阐明MTDH在黑色素瘤Plx4032 耐药中的作用机制，探索LL4a偶联MTDH和/或NRAS siRNA的脂质体系统对抗黑色素瘤Plx4032耐药性的治疗策略。

背景：威罗菲尼已被FDA批准用于治疗BRAF突变的晚期黑素瘤患者。然而，几乎所有的患者在用威罗菲尼治疗后会产生获得性/继发性耐药。因此，探究黑素瘤对威罗菲尼产生耐药的新机制，研发新型针对已产生威罗菲尼耐药黑素瘤的靶向治疗策略，对黑素瘤的治疗具有重大医学意义。.主要研究内容：本课题利用cell-SELEX技术、流式细胞术、共聚焦成像实验等筛选并鉴定黑素瘤威罗菲尼耐药细胞特异性核酸适体；通过流式细胞术、琼脂糖凝胶电泳及裸鼠肿瘤活体成像实验，对核酸适体的特异性与稳定性进行表征；通过aptamer-pull down实验、LC-MS/MS QSTAR分析、western blot、免疫荧光共定位和RNA干扰实验等探寻并鉴定特异性核酸适体的结合靶标；通过免疫共沉淀、western blot、RNA干扰实验和MTT等初步研究黑素瘤对威罗菲尼产生耐药的新机制。.重要结果：本研究成功筛选到能够特异性结合靶细胞Mel28-PLX而不结合对照细胞Mel28的核酸适体LL4，结合Mel28-PLX的解离常数为101.1±16.39 nM。通过对核酸适体LL4进行截短优化，发现截短序列LL4A与靶细胞结合能力更强，解离常数为82.18±12.99 nM，且LL4A在生理条件下具有低细胞毒性和较高稳定性。裸鼠肿瘤活体成像实验发现LL4A能够识别并靶向体内威罗菲尼耐药黑素瘤细胞。LC-MS/MS QSTAR分析发现跨膜蛋白CD63是核酸适体LL4A的结合靶标。CD63/TIMP1/β1-integrin复合物在威罗菲尼耐药黑素瘤细胞中高表达并激活NF-κB和ERK1/2信号通路增强细胞耐药性。.科学意义：本研究首次筛选并鉴定出可以在体外和体内特异性结合黑素瘤威罗菲尼耐药细胞的核酸适体LL4A，且具有较好的血清稳定性、温度稳定性以及低细胞毒性。未来它可以作为预后的潜在成像探针或作为针对已产生威罗菲尼耐药黑素瘤的新型治疗药物的靶向递送载体。同时，我们发现CD63蛋白是核酸适体LL4A的结合靶标，威罗菲尼耐药细胞中CD63/TIMP1/β1-integrin复合物表达升高进而激活NF-κB和ERK1/2信号通路促进黑色素瘤威罗菲尼耐药。因此，我们的结果表明未来可以通过抗体或小分子抑制剂抑制CD63表达或使用LL4A作为CD63 siRNA递送载体来开发新型威罗菲尼联合治疗策略。