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

目的构建人microRNA149(Hsa-miR-149)抑制表达慢病毒载体,并探讨抑制miR-149表达对黑色素瘤Mel-RM细胞增殖及迁移的影响。方法由miRBase及NCBI数据库查找获得miR-149序列,设计合成其引物序列,通过退火反应获得双链DNA寡核苷酸片段,与载体p BS-h U6-1连接,将连接后的载体与FG12连接,经测序鉴定后获得重组慢病毒载体;将重组慢病毒质粒分别与包装辅助质粒共转染至293T细胞中,包装产生病毒,进行病毒滴度测定,再用包装后的病毒感染Mel-RM细胞,利用实时荧光定量PCR法检测miR-149表达水平。同时用MTT法及细胞迁移实验分别检测细胞增殖和迁移能力。结果成功构建了抑制miR-149表达的慢病毒载体,测序证实了所插入的基因序列正确。在荧光显微镜下观察到转染后的297T细胞表达大量绿色荧光蛋白,收集病毒,用梯度滴定法测得的重组载体病毒滴度为3.2×1010TU/L。将病毒感染Mel-RM细胞显示可有效降低miR-149的表达水平(P<0.05)。MTT法结果显示与感染空载FG12病毒的对照组相比,感染抑制miR-149表达重组质粒的实验组中活细胞数明显减少(P<0.05)。细胞迁移实验结果显示与对照组相比实验组明显抑制了Mel-RM细胞的迁移(P<0.001)。结论成功构建抑制miR-149表达的慢病毒载体,抑制miR-149的表达可以抑制Mel-RM细胞的增殖和迁移。并为后续进一步研究miR-149对黑色素瘤细胞发生发展的影响提供依据。

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 293 T 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 293 T packaging cells and melanoma cells expressed green fluorescence. The titer of concentrated virus was3. 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.