反义microRNA-21慢病毒表达载体的构建及对喉癌细胞的增殖抑制作用

Construction and identification of lentiviral vector of anti-miR-21 oligonucleotides and regulation on proliferation of laryngeal squamous cell carcinoma

目的:克隆反义微小RNA hsa-miR-21并构建其慢病毒表达载体,观察反义寡核苷酸重组慢病毒(ASO-miR-21)对喉癌细胞增殖的调控作用。方法:针对hsa-miR-21核苷酸序列,设计并合成mir-21反义寡核苷酸,将pGCSIL-GFP Vector经双酶切后连接产生pGCSIL-GFP-miR-21慢病毒表达载体,PCR筛选阳性克隆,测序鉴定。pGC-LV-miR-21、pHelper 1.0和pHelper 2.0质粒共转染细胞293T,包装慢病毒。以293T细胞绿色荧光蛋白(green fluorescent protein,GFP)的表达水平测定病毒滴度。采用MTT法及克隆形成实验检测反义miR-21(ASO-miR-21)重组慢病毒对喉癌细胞增殖能力的影响。结果:成功构建反义hsa-miR-21的慢病毒表达载体,采用ASO-miR-21抑制喉癌细胞内高水平表达miR-21的活性后,可显著抑制喉癌细胞增殖。结论:成功构建反义miR-21的慢病毒表达载体,ASO-miR-21可以明显抑制喉癌细胞增殖。

Objective：To construct a lentiviral expression vector for antisense oligonucleotid（ASO） has-miR-21 and determine its effect on proliferation of human laryngeal carcinoma cell line.Methods： Towards the miR-21 sequences of human,design and synthesis antisense oligonucleotide miR-21.The recombinant plasmid pGCSIL-GFP-miR-21was confirmed by restriction endonuclease analysis and sequencing.293T cells were cotransfected with the recombinant lentiviral vector together with lentivirus package plasmid mix containing pGC-LV-miR-21,pHelper 1.0 and pHelper 2.0.Virus titer was measured according to the expression level of GFP.MTT assay and colony formation experiment were used to observe the inhibit on percentage of ASO-miR-21 on the proliferation of Hep-2 cells.Results： The lentivirus vector of ASO pGC-LV-miR-21 was successfully constructed and it can inhibit proliferation of Hep-2 cells.Conclusion： The lentivirus vector of ASO-miR-21 was successfully constructed and it can inhibit proliferation of laryngeal carcinoma in vitro.