过表达lncRNA HOTAIR基因的风湿关节炎成纤维样滑膜细胞稳定株构建与筛选

Construction and screening of fibroblast-like synoviocytes in human rheumatoid arthritis stably overexpressing lncRNA HOTAIR

目的 HOTAIR基因与类风湿关节炎(RA)血管翳的形成紧密相关。文中旨在建立并筛选过表达lncRNA HOTAIR的人类风湿关节炎成纤维样滑膜细胞(HFLS-RA)稳定株,为进一步研究lncRNA HOTAIR在RA发病中的作用奠定基础。方法采用PCR扩增lncRNA HOTAIR目的基因,连接到BamHI-HF-HF和XhoI酶切后的PMT406载体上,构建好的质粒经测序鉴定后,转染293T细胞包装慢病毒,分别用重组质粒及空载体慢病毒感染HFLS-RA,构建和筛选稳转株;采用Trizol法分别提取空白细胞、阴性转染细胞和过表达细胞的总RNA后,反转录得到cDNA进行qPCR扩增,根据检测结果进行相对表达量的计算。结果过表达细胞的lncRNA HOTAIR相对表达量(30.329±3.860)较空白细胞和阴性转染细胞(1.001±0.048、0.892±0.247)明显升高,差异有统计学意义(P<0.05);空白细胞和阴性转染细胞差异无统计学意义(P>0.05)。说明过表达lncRNA HOTAIR的HFLS-RA细胞稳定株构建成功。结论成功构建了过表达lncRNA HOTAIR的HFLS-RA细胞稳定株,为进一步探究lncRNA HOTAIR在RA发生发展中的作用提供了实验材料。

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 HO? TAIR. 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),with no statistically significant difference between the latter two groups(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.