microRNA-223过表达与抑制表达 慢病毒载体的构建及鉴定
Construction and identification of recombinant lentivirus vector for microRNA-223 overexpression and suppression
作者：王韵 吉宁 周敏 江潞 陈谦明
Author：Wang Yun, Ji Ning, Zhou Min, Jiang Lu, Chen Qianming.
收稿日期：2015-03-16 年卷（期）页码：2015,33(5):451-451-455
期刊名称：华西口腔医学杂志
Journal Name：West China Journal of Stomatology
关键字：microRNA-223,慢病毒,过表达载体,抑制表达载体,
Key words：micorRNA-223; ,lentivirus vector; ,overexpression vector; ,suppression vector,
基金项目：
国家自然科学基金资助项目（81321002，81270040）；新世纪优秀人才支持计划资助项目（NCET-12-0378）
中文摘要
目的 构建 microRNA-223过表达慢病毒载体及抑制表达慢病毒载体，并验证其过表达或抑制表达microRNA-223的效率。方法 设计针对microRNA-223前体的过表达基因片段及针对microRNA-223成熟体的反义片段，应用聚合酶链反应（PCR）调取目的基因及通过退火反应获得双链DNA寡聚核苷酸片段。并通过基因重组技术将目的 基因片段及反义片段分别克隆到GV229及GV232慢病毒载体中，进行PCR鉴定及DNA测序比对分析，构建相应的 microRNA-223过表达慢病毒载体及抑制表达慢病毒载体。利用荧光定量PCR法检测microRNA-223表达水平。结果 对阳性克隆进行PCR鉴定及DNA测序证明，microRNA-223过表达慢病毒载体及抑制表达慢病毒载体构建成功。microRNA-223过表达慢病毒载体能显著提高细胞中microRNA-223表达水平，microRNA-223抑制表达慢病毒载体能明显抑制细胞中microRNA-223表达水平。结论 成功构建microRNA-223过表达慢病毒载体及抑制表达慢病毒载体，为进一步研究microRNA-223对口腔癌发生发展的影响及其机制提供了稳定的细胞转染载体。
英文摘要
Objective To construct microRNA-223 overexpression and suppression lentivirus vectors and determine their effects after infecting oral squamous cell carcinoma (OSCC) cell line. Methods Lentivirus vectors GV229 and GV232 were cut by the restriction sites of AgeⅠ and EcoRⅠ and connected to the target gene, which contained mature microRNA-223 and microRNA-223 oligonucleotide. Real-time polymerase chain reaction (PCR) method was used to detect the microRNA-223 expression level after infecting the recombinant lentivirus vector into the OSCC cell line. Results The successful construction of microRNA-223 recombinant lentivirus vectors was confirmed by the PCR method and DNA sequencing. HN-30 cell infected with microRNA-223 overexpression vector showed a significant increased in microRNA-223 expression, whereas HN-30 cell infected with microRNA-223 inhibitor vector suppressed microRNA-223 expression. Conclusion The microRNA-223 overexpression and suppression lentivirus vectors are successfully constructed. These vectors could alter the expression level of microRNA-223 in OSCC cell line significantly, and provide a stable cell line for functional studies in the future.
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