Construction and screening of fibroblast-like synoviocytes in human rheumatoid arthritis stably overexpressing lncRNA HOTAIR / 医学研究生学报

ABSTRACT

Objective　The HOTAIR gene is closely related to pannus formation in rheumatoid arthritis (RA). This study aimed to construct and screen fibroblast-like synoviocytes in human RA (HFLS-RA) stably overexpressing lncRNA HOTAIR, and to pave the way for further study of the role of lncRNA HOTAIR in the pathogenesis of RA.　Methods　LncRNA HOTAIR was cloned and linked to the PMT406 vector digested by BamHI-HF-HF and XhoI. The constructed plasmids were sequenced, identified and then transfected into 293T cells to pack lentivirus. The HFLS-RA cells were infected with the recombinant and empty vector lentiviruses, and purinomycin was employed to screen the lncRNA HOTAIR-overexpressed and control cell lines. The total RNA was extracted from the blank, negatively transfected and overexpressed cells by Trizol, and the cDNA obtained by reverse transcription was amplified by qPCR, followed by determination of the expression of lncRNA HOTAIR.　Results　The relative expression of lncRNA HOTAIR was significantly higher in the overexpression group than in the blank control and negative transfection groups (30.329 ± 3.860 vs 1.001 ± 0.048 and 0.892 ± 0.247, P 0.05).　Conclusion　The HFLS-RA cell line stably overexpressing lncRNA HOTAIR was successfully constructed, which has provided some experimental evidence for further investigation of the role of lncRNA HOTAIR in the pathogenesis of RA.