^(99)Tc^(m)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^(99)Tc^(m)labeled IncRNA HOTAIR antisense probe and its effect on the activity of human glioma U87 cells

作　　者：任炯羽 张熹元 张福禄 冯薛烟 赵倩[1] Ren Jiongyu;Zhang Xiyuan;Zhang Fulu;Feng Xueyan;Zhao Qian(Department of Nuclear Medicine,General Hospital of Ningxia Medical University,Yinchuan 750004,China;Xiangya School of Medicine,Central South University,Changsha 410006,China;Graduate School of Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学总医院核医学科,银川750004 [2]中南大学湘雅医学院,长沙410006 [3]宁夏医科大学研究生院,银川750004

出　　处：《国际放射医学核医学杂志》2021年第3期162-169,共8页International Journal of Radiation Medicine and Nuclear Medicine

基　　金：国家自然科学基金(81560292)。

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^(99)Tc^(m)-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^(99)Tc^(m)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-^(99)Tc^(m)-HYNIC-ASON组(转染组)和^(99)Tc^(m)-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-^(99)Tc^(m)-HYNIC-ASON组(转染组)、^(99)Tc^(m)-HYNIC-ASON组(未转染组)、^(99)Tc^(m)-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^(99)Tc^(m)-HYNIC-ASON的标记率为(90.0±5.6)%。琼脂糖凝胶电泳结果显示,^(99)Tc^(m)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-^(99)Tc^(m)-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-^(99)Tc^(m)-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-^(99)Tc^(m)-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�Objective To prepare a novel antisense oligonucleotides(ASON)probe,namely,^(99)Tc^(m)-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^(99)Tc^(m).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-^(99)Tc^(m)-HYNIC-ASON(transfection group)and^(99)Tc^(m)-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-^(99)Tc^(m)-HYNIC-ASON(transfection group),^(99)Tc^(m)-HYNIC-ASON(non-transfection group),and^(99)Tc^(m)-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^(99)Tc^(m)-HYNIC-ASON was(90.0±5.6)%.Gel electrophoresis results confirmed that^(99)Tc^(m)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-^(99)Tc^(m)-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-^(99)Tc^(m)-HY

关 键 词：RNA 长链非编码 寡核苷酸类 反义 神经胶质瘤 99m锝高锝酸钠 分子探针 同源异型盒基因转录的反义基因间RNA 

分 类 号：R730.44[医药卫生—肿瘤] R739.41[医药卫生—临床医学]