核酸适体RCC-H1b对肾癌识别和细胞核定位的研究

Cancer is a serious worldwide human disease. In order to obtain an novel aptamer for the specific recognition of renal cell carcinoma, we synthesized a "swan" library, and applied flow cytometry experiments with the human renal clear cell carcinoma cell line 786-O as the target cell and the human renal fibroblast cell line KFB as the control cell. We found that RCC-H1 specifically binds to 786-O, and its optimized sequence RCC-H1b displays a nuclear localization. In order to investigate the recognition properties of RCC-H1b in complex biological environment, we also tested its binding abilities to clinicopathological paraffin tissue microarray and mouse RCC- xenografted tumor and found that it can identify both specifically. At present, the tumor biomarkers of renal cell carcinoma are few, non-specific, and not widely used, so it is necessary to find new molecular probes and tumor biomarkers. With these technologies, including LC-MS/MS, SERS, shRNA lentivirus, flow cytometry, SDS-PAGE, western blot and so on, we plan to identify the target protein that RCC-H1b binds to and further explore the molecular mechanism of RCC-H1b nuclear localization.

癌症是全球范围内严重危害人类健康的疾病。为了获得特异性识别肾癌的核酸适体，我们合成了swan文库，以人肾透明细胞癌细胞系786-O为靶细胞，以人肾成纤维细胞系KFB为对照细胞，进行流式结合验证。我们发现RCC-H1能特异性结合786-O，序列优化后的RCC-H1b具有核定位现象。为了考察RCC-H1b在复杂生物环境下的识别性能，我们表征了RCC-H1b分别与临床病理石蜡组织芯片和小鼠活体肿瘤的结合情况，发现它对病人癌组织和裸鼠RCC移植瘤都能保持良好的识别能力。目前肾癌的肿瘤标志物较少，无特异性，且用途不广，因此发现肾癌新的分子探针和肿瘤标志物十分有必要，我们拟通过生物质谱、SERS、shRNA慢病毒、流式细胞术、SDS-PAGE、western blot等技术开展研究，初步鉴定RCC-H1b在肾透明细胞癌中结合的蛋白靶标，及探讨RCC-H1b细胞核定位的分子机制。

肾癌是一种常见的恶性肿瘤，由于缺乏特定的肿瘤标记物，主要采用影像学检查进行肾癌诊断。我们通过将肾癌786-O细胞与swan文库共孵育，前期已经筛选出可以特异性结合肾癌细胞的核酸适体RCC-H1b。我们发现核酸适体具有明显核定位现象，并且能抑制细胞周期和促进细胞凋亡，见已发表论文。首先通过流式细胞术考察RCC-H1b与不同类型内吞抑制剂处理786-O细胞的流式结合情况，我们初步判断RCC-H1b 结合靶标类型为蛋白，并且可能通过细胞松弛素D和M-b-CD介导内吞作用。我们进一步通过Aptamer pulldown实验核酸适体RCC-1b与786-O细胞潜在的结合蛋白，然后进行质谱分析和确认出差异条带nap1l4蛋白。最后通过慢病毒干扰实验进行验证靶蛋白。本课题将实现加对核酸适体RCC-H1b在786-O中的结合靶标和入核机制的深入理解，大力推进加核酸适体RCC-H1b在肾透明细胞癌的识别和治疗的应用，并将为核酸适体的生物功能研究这个方向注入创新动力。