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In Vitro Research on MicroRNA-155 Targeted Radiolabeled Oligonucleotide in Breast Cancer Cells

HUO Yan;KANG Lei;WANG Rong-fu;PANG Xiao-xi;LIU Min;YAN Ping;ZHANG Chun-li   

  1. Department of Nuclear Medicine, Peking University First Hospital, Beijing 100034, China
  • Online:2017-05-20 Published:2017-05-22

靶向微小RNA-155的放射性探针在乳腺肿瘤细胞的靶向研究

霍焱;康磊;王荣福;庞小溪;刘敏;闫平;张春丽   

  1. 北京大学第一医院 核医学科,北京100034

Abstract:

To investigate the role of regulation, uptake and distribution of miR-155 targeted radiolabeled probes in MCF-7 cell line and to provide an effective candidate probe for future in vivo studies. MiR-155 targeted or nonsense control probe was radiolabeled with technetium-99m (99mTc) using bifunctional chelator NHS-MAG3. The serum stability was evaluated by the Mini-Scan thin layer chromatography. After transfected in MCF-7 cells, miR-155 targeted probe was evaluated by western blotting. Its cellular uptake was measured at different time points. Further, the distribution of fluorescent protein labeled FAM and nonsense probes were evaluated in MCF-7 cells by confocal microscopy. The radiolabeled efficiency of 99mTc-labeled miR-155 targeted probe was 97%, radiochemical purity was greater than 98%, and radioactive specific activity was 3.75 GBq/μg. In fresh human serum for 12 h, its radiochemical purity was greater than 95%. Compared with untreated cells, unlabeled and labeled miR-155 targeted probes up-regulated the expression of C/EBPβ protein in MCF-7 cells. The cellular uptake of 99mTc-labeled miR-155 targeted probe was significantly higher than that of untreated cells in MCF-7 tumor cells. Furthermore, the fluorescence-labeled miR-155 targeted probe showed a stable and efficient targeting distribution in MCF-7 cells at 24 h. Therefore, miR-155 targeted radiolabeled probe has good targeting and biological activity at the cellular level in MCF-7 cells, and has potential application value for in vivo future imaging.

Key words: MicroRNA, radiolabel, cellular uptake, fluorescent imaging, MCF-7

摘要:

探讨靶向microR-155(微小RNA-155,miR-155)放射性探针在乳腺癌细胞水平的靶向调控、摄取和分布情况,以期为活体靶向研究提供有效的候选探针。通过双功能偶联剂NHS-MAG3构建放射性99mTc标记的miR-155靶向和无义探针。置于人新鲜血清中,使用薄层层析法测定其放化纯度。转染乳腺癌MCF-7细胞后,通过western blotting评价其对miR-155靶蛋白C/EBPβ的调节作用,并测定不同时间点该探针的细胞摄取率。合成荧光蛋白FAM标记的靶向和无义探针,通过共聚焦显微镜评价荧光标记探针在肿瘤细胞内的分布情况。结果显示,99mTc标记miR-155靶向探针的标记率为97%,放化纯度大于98%,放射性比活度为3.75 GBq/μg。在人新鲜血清中12 h,放化纯度大于95%。与未处理细胞对比,未标记和标记的miR-155靶向探针均能上调MCF-7细胞内C/EBPβ蛋白表达水平。随着孵育时间延长,99mTc标记的miR-155靶向探针在MCF-7肿瘤细胞的细胞摄取率明显高于对照组。荧光标记的miR-155靶向探针在MCF-7细胞中24 h显示出稳定、高效的靶向分布。结果表明,miR-155靶向的放射性探针在乳腺癌细胞水平具有良好的靶向性和生物活性,具有潜在的体内肿瘤显像应用价值。

关键词: 微小RNA, 放射性标记, 细胞摄取, 荧光成像, MCF-7