韩康, 赵廷宝, 卞娜, 蔡成魁, 颜世举, 王鑫, 马琼, 沙浩, 董川, 杨彤涛, 周勇, 马保安. miR-194慢病毒表达质粒的构建及对人骨肉瘤细胞系U2-OS转移的相关研究[J]. 中国肿瘤临床, 2014, 41(12): 753-757. DOI: 10.3969/j.issn.1000-8179.20140226
引用本文: 韩康, 赵廷宝, 卞娜, 蔡成魁, 颜世举, 王鑫, 马琼, 沙浩, 董川, 杨彤涛, 周勇, 马保安. miR-194慢病毒表达质粒的构建及对人骨肉瘤细胞系U2-OS转移的相关研究[J]. 中国肿瘤临床, 2014, 41(12): 753-757. DOI: 10.3969/j.issn.1000-8179.20140226
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HAN Kang, ZHAO Tingbao, BIAN Na, CAI Chengkui, YAN Shiju, WANG Xin, MA Qiong, SHA Hao, DONG Chuan, YANG Tongtao, ZHOU Yong, MA Bao'an. Effects of MiR-194 on the metastasis of human osteosarcoma cell line U2-OS by recombinant lentivirus vector[J]. CHINESE JOURNAL OF CLINICAL ONCOLOGY, 2014, 41(12): 753-757. DOI: 10.3969/j.issn.1000-8179.20140226
Citation: HAN Kang, ZHAO Tingbao, BIAN Na, CAI Chengkui, YAN Shiju, WANG Xin, MA Qiong, SHA Hao, DONG Chuan, YANG Tongtao, ZHOU Yong, MA Bao'an. Effects of MiR-194 on the metastasis of human osteosarcoma cell line U2-OS by recombinant lentivirus vector[J]. CHINESE JOURNAL OF CLINICAL ONCOLOGY, 2014, 41(12): 753-757. DOI: 10.3969/j.issn.1000-8179.20140226
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miR-194慢病毒表达质粒的构建及对人骨肉瘤细胞系U2-OS转移的相关研究

Effects of MiR-194 on the metastasis of human osteosarcoma cell line U2-OS by recombinant lentivirus vector

    摘要
  • 摘要:
      目的   通过构建携带针对miR-194的慢病毒沉默及过表达载体，研究miR-194对骨肉瘤细胞转移的影响。
      方法   以miR-194前体及成熟体序列为模板，设计引物，进行PCR扩增，酶切后克隆于慢病毒载体plent-i GFP中，转染293T细胞，包装慢病毒颗粒，检测滴度，最终对人骨肉瘤细胞系U2-OS进行感染。获取稳定感染细胞后，进行Transwell、划痕等实验。
      结果   1）慢病毒表达质粒构建成功，沉默表达及过表达重组慢病毒的滴度分别为4×108TU/mL及1.5×108TU/mL；2）稳定过表达miR-194的人骨肉瘤细胞系U2-OS的转移能力较其他组有明显降低（P < 0.01）。
      结论   成功构建了miR-194慢病毒表达载体，miR-194的表达水平对U2-OS细胞的转移能力具有显著影响，miR-194可能成为骨肉瘤治疗的潜在新靶点。

     

    Abstract:
      Objective   This study aimed to construct a lentiviral expression vector for microRNA-194 and investigate its effect on the metastasis of human osteosarcoma cell line U2-OS.
      Methods   Pri- and mature miR-194 amplified by PCR were inserted into the plenty-GFP vector and identified by restriction endonuclease digestion and nucleotide sequencing. The osteosarcoma cell line U2-OS was transfected with the lentivirus. Then, the stable transfected cells were used in Transwell and wound healing assay.
      Results   Restriction analysis and sequencing showed that the recombinant lentiviral expression vector was constructed correctly. The titers of obtained overexpression and suppression expression recombinant lentivirus were 1.5*108 and 4*108 TU/ml. Cell metastasis ability was significantly different in different experimental groups (P < 0.01).
      Conclusion   The lentiviral expression vector for microRNA-194 was successfully constructed. MicroRNA-194 could influence the metastasis of the osteosarcoma cell line U2-OS; thus, it could be further explored as a potential target in osteosarcoma therapy.

     

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