〔Abstract〕 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.