安徽医科大学学报2017，Vol.52Issue(12)：1756-1763,8.DOI:10.19405/j.cnki.issn1000-1492.2017.12.003
人microRNA149抑制表达慢病毒载体的构建及对黑色素瘤细胞增殖及迁移的影响
Construction of miR-149 suppression lentivirus vector and effect of low miR-149 expression on proliferation and migration of melanoma cells
摘要
Abstract
Objective To construct miR-149 suppression lentivirus vector and investigate the effect of low miR-149 expression on proliferation and migration of melanoma cells.Methods The miR-149 sequence was obtained from the miRBase and NCBI databases,the pair of primers were designed and synthesized.Double-stranded DNA oligonucleotide fragments were obtained by annealing response,the target gene was ligated with vector FG12,the lentiviral plasmid was identified by double enzyme digestion and sequencing analysis.Lentivirus was generated by cotransfection with the above construct with packaging plasmids into 293T cell,lentivirus was harvested,the virus drops were measured.Lentivirus were used to infect Mel-RM cells,then real-time polymerase chain reaction(PCR)method was used to detect the miR-149 expression level.MTT assay was used to examine the cell proliferating capacity,cell migration assay was used to examine the cell migration.Results The successful construction of miR-149 recombinant lentivirus vectors was confirmed by DNA sequencing.Fluorescence microscope observation showed that the 293T packaging cells and melanoma cells expressed green fluorescence.The titer of concentrated virus was 3.2 × 1010 TU/ml.Melanoma cells infected with lentiviruses,the expression level of miR-149 reduced effectively (P ＜ 0.05).MTT assay results showed that compared with the control group,experimental group cell proliferate more slowly(P ＜ 0.05).The migration results showed that experimental group cell migrated more slowly (P ＜0.001).Conclusion miR-149 suppression lentivirus vectors is successfully constructed.Low miR-149 expression can suppress proliferation and migration of melanoma cells,and provide a basis for further study of the effect of miR-149 on the development of melanoma cells.关键词
miR-149/慢病毒载体/增殖/迁移/Mel-RM细胞Key words
miR-149/lentivirus vector/proliferation/migration/melanoma cell分类
医药卫生引用本文复制引用
王佩佩,王道月,轩菡,陈振东,缪琳..人microRNA149抑制表达慢病毒载体的构建及对黑色素瘤细胞增殖及迁移的影响[J].安徽医科大学学报,2017,52(12):1756-1763,8.基金项目
国家自然科学基金(编号:81101525) （编号:81101525）
