HOTAIR过表达慢病毒载体的构建及稳定细胞株的建立

Construction of HOTAIR overexpression lentivirus vector and establishment of stable cell lines

目的构建稳定过表达HOTAIR的人滋养细胞HTR-8/SVneo细胞系,并验证HOTAIR的表达。方法利用PCR技术扩增HOTAIR基因全长序列,并将其克隆入LV5载体中,构建LV5-HOTAIR载体,重组质粒经双酶切鉴定及测序验证成功后,转染人胚肾细胞293T,包装过表达病毒颗粒,制备并浓缩慢病毒颗粒。将构建好的过表达HOTAIR慢病毒载体感染HTR-8/SVneo细胞,采用嘌呤霉素筛选出HOTAIR稳定表达的细胞系,荧光显微镜观察绿色荧光表达;qRT-PCR验证HOTAIR表达。结果重组质粒酶切得到的条带与预期相符,过表达HOTAIR慢病毒载体中序列与目标序列一致。荧光显微镜下过表达HOTAIR组细胞有绿色荧光表达;qRT-PCR结果表明过表达HOTAIR的HTR-8/Svneo稳定细胞株中HOTAIR表达是Control组的206.3倍(P<0.05),是NC组的232.8倍(P<0.05)。结论成功建立稳定过表达HOTAIR的HTR-8/SVneo细胞,且过表达HOTAIR的HTR-8/Svneo稳定细胞株中HOTAIR mRNA表达水平明显升高。

Objective To establish a stable human trophoblast cell line HTR-8/SVneo with overexpression of HOTAIR and to verify the expression of HOTAIR. Methods PCR was used to amplify the full length sequence of HOTAIR gene and clone it into LV5 vector. LV5-HOTAIR vector was constructed. The recombinant plasmid was identified by double enzyme digestion and verified by sequencing. The constructed overexpressed HOTAIR lentivirus vector was infected with HTR-8/SVneo cells,and the cell lines with stable expression of HOTAIR were screened by purinomycin,and the level of GFP expression was observed by fluorescence microscope. qRT-PCR verified the expression of HOTAIR. Results The bands obtained by recombinant plasmid digestion were consistent with the expectation,and the sequences of overexpressed HOTAIR lentivirus vectors were consistent with the target sequences.Under fluorescence microscope,the cells in the overexpressed HOTAIR group showed green fluorescence expression. qRT-PCR results showed that the HOTAIR expression in HTR-8/Svneo stable cell lines with overexpression of HOTAIR was 206. 3 times higher than that in the Control group( P < 0. 05) and 232. 8 times higher than that in the NC group( P < 0. 05). Conclusion The stable HOTAIR-overexpressed HTR-8/SVneo cells were successfully established,and the expression level of HOTAIR mRNA significantly increased in the stable HOTAIR-overexpressed HTR-8/SVneo cell lines.