99Tcm标记lncRNA HOTAIR反义寡核苷酸探针的制备及其对人脑胶质瘤U87细胞活性的影响

Preparation of 99Tcm labeled lncRNA HOTAIR antisense probe and its effect on the activity of human glioma U87 cells

    摘要
  • 摘要:
    目的 制备新型的靶向长链非编码RNA（lncRNA）同源异型盒基因转录的反义基因间RNA（HOTAIR）的反义寡核苷酸（ASON）探针99Tcm-HYNIC-ASON（HYNIC为联肼尼克酰胺），探讨其对人脑胶质瘤U87细胞增殖和迁移能力的影响。
    方法 设计并通过化学修饰合成HOTAIR的ASON，使用双功能螯合剂HYNIC偶联99Tcm并进行纯化。采用快速薄层层析(ITLC)法和琼脂糖凝胶电泳分别检测探针的标记率、放射化学纯度、体外稳定性及完整性。细胞摄取实验分为2组：Lipo-99Tcm-HYNIC-ASON组（转染组）和99Tcm-HYNIC-ASON组（未转染组），通过脂质体转染探针，测定人脑胶质瘤U87细胞对探针的摄取率；细胞计数试剂盒8（CCK-8）实验和细胞划痕实验分为3组：Lipo-99Tcm-HYNIC-ASON组（转染组）、99Tcm-HYNIC-ASON组（未转染组）、99Tcm-Control组（对照组），分别检测转染探针后细胞增殖和迁移能力的变化。2组间比较采用Student t检验，多组间比较采用单因素方差分析。
    结果 99Tcm-HYNIC-ASON的标记率为（90.0±5.6）%。琼脂糖凝胶电泳结果显示，99Tcm与探针成功标记并且没有明显的降解，探针孵育12 h的放射化学纯度>80%。细胞摄取实验结果显示，转染后5 h，探针Lipo-99Tcm-HYNIC-ASON在人脑胶质瘤U87细胞中的摄取率最大（0.70%），与未转染组（0.16%）相比，差异有统计学意义（t=17.81，P<0.01）。CCK-8实验结果显示，转染探针Lipo-99Tcm-HYNIC-ASON能抑制人脑胶质瘤U87细胞的增殖能力，与未转染组相比，在各个时间点（1、2、3、4、5 d）的差异均有统计学意义（t=2.336~30.230, 均P<0.05）。细胞划痕实验结果显示，转染探针Lipo-99Tcm-HYNIC-ASON能抑制人脑胶质瘤U87细胞的迁移，3组细胞间隙融合率的差异有统计学意义(F=331.8，P<0.01)，与未转染组相比，转染组细胞间隙融合率明显降低，且差异有统计学意义（60.0%对23.6%， t=51.54，P<0.01）。
    结论 成功合成了靶向人脑胶质瘤lncRNA HOTAIR的探针99Tcm-HYNIC-ASON，该探针具有良好的体外稳定性和靶向结合能力，能够抑制人脑胶质瘤U87细胞的增殖和迁移。

     

    Abstract:
    Objective To prepare a novel antisense oligonucleotides (ASON) probe, namely, 99Tcm-HYNIC-ASON (hydrazine nicotinamide (HYNIC)), targeting long non-coding RNA (lncRNA) homeobox gene anti-sense intergenic RNA (HOTAIR); and explore its effect on the proliferation and migration of human glioma U87 cells.
    Methods HOTAIR ASON was designed and synthesized by chemical modification, and the bifunctional chelating agent (HYNIC) was coupled with 99Tcm. Sephadex G25 was selected for separation and purification. The labeling rate, radiochemical purity, in vitro stability, and integrity of the probe were detected by instant thin-layer chromatography and agarose gel electrophoresis. Human glioma U87 cells were cultured for experimental use. The cell uptake assay was divided into two groups: Lipo-99Tcm-HYNIC-ASON (transfection group) and 99Tcm-HYNIC-ASON (non-transfection group). The probe was transfected by liposome to determine the probe uptake rate of human glioma U87 tumor cells. The cell counting kit-8 (CCK-8) assay and cell scratch assay were divided into three groups, namely, Lipo-99Tcm-HYNIC-ASON (transfection group), 99Tcm-HYNIC-ASON (non-transfection group), and 99Tcm-Control (control group), to detect the changes of cell proliferation and migration after transfection of probe. Student t-test was used for comparison between two groups, and one-way analysis of variance was used for multi-group comparison.
    Results The labeling rate of 99Tcm-HYNIC-ASON was (90.0±5.6)%. Gel electrophoresis results confirmed that 99Tcm and the probe were successfully labeled without evident degradation; the probe showed good stability and radiochemical purity >80% after being incubated for 12 h. The results of cell uptake assay showed that 5 h after liposome transfection, the maximum uptake rate of probe Lipo-99Tcm-HYNIC-ASON in human glioma U87 cells was 0.70%, which was significantly higher than that in the non-transfection group (0.16%; t=17.81, P<0.01). The results of CCK-8 assay showed that the transfection probe (Lipo-99Tcm-HYNIC-ASON) could inhibit the proliferation of human glioma U87 cells, and a significant difference was observed compared with the non-transfection group at 1, 2, 3, 4, 5 d (t=2.336–30.230, all P<0.05). The results of cell scratch assay showed that the transfection probe (Lipo-99Tcm-HYNIC-ASON) could inhibit the migration of human glioma U87 cells, and a significant difference was found in the intercellular fusion rate among the three groups (F=331.8, P<0.01). Compared with the non-transfection group (60.0%), the intercellular fusion rate in the transfection group was significantly lower (23.6%), and the difference was statistically significant (t=51.54, P<0.01).
    Conclusions The ASON probe targeting human glioma lncRNA HOTAIR has been successfully synthesized. The probe has good stability and targeted binding ability in vitro, which can inhibit the proliferation and migration of human glioma U87 cells.

     

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