靶向微小RNA-155的放射性探针在乳腺肿瘤细胞的靶向研究

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

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^(99m)Tc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,^(99m)Tc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3.75GBq/μg。在人新鲜血清中12h,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,^(99m)Tc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24h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。

To investigate the role of regulation,uptake and distribution of miR-155 targeted radiolabeled probes in MCF-7cell 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 sta-bility was evaluated by the Mini-Scan thin layer chromatography.After transfected in MCF-7cells,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-7cells by confocal microscopy.The radiolabeled efficiency of 99mTc-labeled miR-155 targeted probe was97%,radiochemical purity was greater than 98%,and radioactive specific activity was3.75GBq/μg.In fresh human serum for 12 h,its radiochemical purity was greater than95%.Compared with untreated cells,unlabeled and labeled miR-155 targeted probes up-regulated the expression of C/EBPβprotein in MCF-7cells.The cellular uptake of99mTc-labeled miR-155 targeted probe was significantly higher than that of untreated cells in MCF-7tumor cells.Furthermore,the fluorescence-labeled miR-155 targeted probe showed a stable and efficient targeting distribution in MCF-7cells at 24 h.Therefore,miR-155 targeted radiolabeled probe has good targeting and biological activity at the cellular level in MCF-7cells,and has potential application value for in vivo future imaging.