miR-513a-5p慢病毒载体的构建及其对人骨肉瘤细胞放疗敏感性的影响  

Construction of miR-513a-5p lentivirus vector and its effect on radiosensitivity of human osteosarcoma cells

作　　者：代晓燕 赵晓龙 李崇义 杨宇馨 戴楠 DAI Xiaoyan;ZHAO Xiaolong;LI Chongyi;YANG Yuxin;DAI Nan(Department of Oncology,Daping Hospital,Army Medical University,Chongqing 400042,China)

机构地区：[1]陆军军医大学大坪医院肿瘤科,重庆400042

出　　处：《现代肿瘤医学》2020年第21期3657-3661,共5页Journal of Modern Oncology

基　　金：国家自然科学基金青年基金项目(编号:81001000,81101993)。

摘　　要：目的:构建miR-513a-5p慢病毒过表达载体,转染人骨肉瘤细胞株,观察miR-513a-5p对人骨肉瘤细胞放疗敏感性的影响。方法:PCR法扩增人miR-513a-5p基因,克隆入pLentis-CMV-GFP-MCS-PGK-PURO载体获得重组质粒pLentis-miR513a,双酶切鉴定并测序后将正确的重组质粒和对照质粒转染293FT细胞制备慢病毒,分别转染骨肉瘤HOS和U2OS细胞,qRT-PCR法及荧光显微镜鉴定转染结果。克隆形成实验、MTT法检测miR-513a-5p高表达HOS和U2OS细胞在X射线照射下细胞存活情况。结果:双酶切及测序结果确定成功构建miR-513a-5p慢病毒载体pLentis-miR513a。qRT-PCR结果提示,转染骨肉瘤细胞株后miR-513a-5p表达显著升高。克隆形成实验结果显示miR-513a-5p高表达后骨肉瘤细胞在X射线照射下细胞增殖减慢。MTT结果提示miR-513a-5p高表达骨肉瘤细胞经X射线照射后细胞存活减少。结论:成功构建了miR-513a-5p慢病毒载体,建立了高效稳定表达miR-513a-5p的骨肉瘤细胞株,高表达miR-513a-5p能显著增加X射线照射后骨肉瘤细胞的放疗敏感性。Objective:To construct lentivirus vector containing miR-513a-5p gene,and explore the influence of miR-513a-5p on radiosensitivity of osteosarcoma cell line.Methods:Human miR-513a-5p gene was amplified by PCR,cloned into the vector of pLentis-CMV-GFP-MCS-PGK-PURO to obtain the recombinant plasmid pLentis-miR513a.After double enzyme digestion and sequencing,the correct recombinant plasmid and the control plasmid were transfected into 293FT cells to prepare lentivirus,and then transfected into osteosarcoma HOS and U2OS cells,respectively.The transfection results were identified by qRT-PCR and fluorescence microscopy.Clony formation assay and MTT assay were used to detect the survival of HOS and U2OS cells with high expression of miR-513a-5p under X-ray irradiation.Results:The sequencing result proved the sequence of miR-513a-5p in plasmid vector was correct.The results of qRT-PCR indicated that the expression of miR-513a-5p increased significantly after transfection of osteosarcoma cell lines.The result of clony formation and MTT method suggested the osteosarcoma cell lines had slowed proliferation with infection miR-513a-5p lentivirus under X-ray treatment.Conclusion:The miR-513a-5p lentivirus vector was constructed successfully.High expression of miR-513a-5p can significantly increase the radiosensitivity of osteosarcoma cells after X-ray irradiation.

关 键 词：miR-513a-5p 骨肉瘤 慢病毒表达载体 放疗敏感性 

分 类 号：R738.1[医药卫生—肿瘤]