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

发布时间：2019-01-04 07:52

【摘要】：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(P0.05)。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(P0.05)。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(P0.001)。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。
[Abstract]:Objective to construct a human microRNA149 (Hsa-miR-149) vector that inhibits the expression of lentivirus and to investigate the effect of inhibiting miR-149 expression on the proliferation and migration of melanoma Mel-RM cells. Methods the miR-149 sequence was obtained from miRBase and NCBI database, the primer sequence was designed and synthesized, the double-stranded DNA oligonucleotide fragment was obtained by annealing reaction, and ligated with the vector p BS-h U6-1. The ligated vector was connected with FG12. The recombinant lentivirus vector was obtained by sequencing. The recombinant lentivirus plasmids were cotransfected into 293T cells respectively with packaging assistant plasmids. The virus was packaged to produce the virus, then the virus titer was determined, and then the packaged virus was used to infect the Mel-RM cells. The expression of miR-149 was detected by real-time fluorescence quantitative PCR. At the same time, MTT assay and cell migration assay were used to detect cell proliferation and migration ability. Results Lentivirus vector was successfully constructed to inhibit the expression of miR-149, and the sequence of the inserted gene was confirmed by sequencing. A large amount of green fluorescent protein was expressed in 297T cells transfected with fluorescence microscope. The viral titer of the recombinant vector was 3.2 脳 10 ~ (10) TU / L measured by gradient titration. The expression level of miR-149 was significantly decreased by Mel-RM cells infected with virus (P0.05). MTT results showed that compared with the control group infected with non-loaded FG12 virus, the expression of miR-149 was significantly lower than that of the control group infected with non-loaded FG12 virus. The number of living cells in the experimental group infected with the recombinant plasmid expressing miR-149 was significantly decreased (P0.05). The results of cell migration assay showed that the migration of Mel-RM cells was significantly inhibited in the experimental group compared with the control group (P0. 001). Conclusion Lentivirus vector was successfully constructed to inhibit the expression of miR-149 and the expression of miR-149 could inhibit the proliferation and migration of Mel-RM cells. It provides the basis for further study on the effect of miR-149 on the development of melanoma cells.
【作者单位】：安徽医科大学第二附属医院肿瘤科;安徽医科大学第二附属医院科研部;
【基金】：国家自然科学基金(编号:81101525)
【分类号】：R739.5

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