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VASN核酸适配体与端粒酶逆转录酶脱氧核酶偶联物的生物学功能研究

Functional Study of Conjugate of VASN Aptamer-DNAzyme for Telomerase Reverse Transcriptase
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摘要 目的研究肝癌细胞特异性膜蛋白VASN(vasorin)的核酸适配体V4-2与靶向端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)的脱氧核酶(DNAzyme,Dz)偶联形成的偶联物V4-2-Dz在体外和肝癌细胞内的靶RNA切割活性,为脱氧核酶递送提供新策略。方法Mfold网站预测V4-2-Dz的二级结构,通过体外切割实验检测V4-2-Dz的切割活性,通过流式细胞术和激光共聚焦实验检测V4-2-Dz和肝癌细胞HepG2结合的能力,利用RT-qPCR和MTT法分析V4-2-Dz作用于HepG2细胞对h TERT mRNA表达水平和细胞增殖的影响。结果体外切割实验表明,V4-2-Dz具有切割hTERT RNA活性;流式细胞术和激光共聚焦实验结果显示,V4-2-Dz可以特异结合并进入HepG2细胞,V4-2-Dz可显著降低HepG2细胞中hTERT mRNA水平并抑制细胞增殖。结论VASN蛋白的核酸适配体与端粒酶逆转录酶的脱氧核酶偶联物V4-2-Dz具有靶向肝癌细胞递送和切割活性,基于适配体和脱氧核酶的偶联物为肿瘤药物研究提供新的思路。 Objective To investigate the target RNA cleavage activity of V4-2-Dz,a conjugate formed by coupling the aptamer V4-2 of the hepatocellular carcinoma cell-specific membrane protein VASN(vasorin)with the DNAzyme(Dz)targeting the telomerase reverse transcriptase(hTERT),in vitro and in hepatocellular carcinoma cells,and to establish a new strategy for DNAzyme delivery.Methods The secondary structure of V4-2-Dz was predicted by using the Mfold website and the cleavage activity of V4-2-Dz was examined by in vitro cleavage assay.And then,the ability of V4-2-Dz binding to hepatocellular carcinoma HepG2 cells was examined by flow cytometry and laser confocal assay.The effects of V4-2-Dz on the hTERT mRNA expression and cell proliferation of HepG2 cells were analyzed by using RT-qPCR and MTT assays.Results In vitro cleavage assays showed that V4-2-Dz has hTERT RNA cleavage activity.Flow cytometry and laser confocal results showed that V4-2-Dz specifically binds to HepG2 cells and could reduce the hTERT mRNA levels and inhibit cell proliferation significantly.Conclusion The conjugate of aptamer V4-2 with DNAzyme of telomerase reverse transcriptase,V4-2-Dz,has targeted cellular delivery and cleavage activity.The conjugate based on the aptamer and DNazyme provides a new idea for antitumor drug research.
作者 马斐越 李慧 刘雪梅 台福敏 邵宁生 高波 郑晓飞 MA Fei-Yue;LI Hui;LIU Xue-Mei;TAI Fu-Min;SHAO Ning-Sheng;GAO Bo;ZHENG Xiao-Fei(Beijing Key Laboratory for Radiobiology,Institute of Radiation Medicine,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China;Institute of Military Cognition and Brain Sciences,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China)
出处 《生物化学与生物物理进展》 SCIE CAS CSCD 北大核心 2023年第9期2175-2184,共10页 Progress In Biochemistry and Biophysics
基金 国家自然科学基金(31570817,32071290)资助项目。
关键词 VASN蛋白 适配体 端粒酶逆转录酶 脱氧核酶 肝癌 VASN protein aptamer telomerase reverse transcriptase DNAzyme hepatoma
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