长链非编码RNA GAPLINC对类风湿关节炎成纤维样滑膜细胞增殖表型的影响

Effect of LncRNA GAPLINC on the Cell Proliferation of Rheumatoid Arthritis Fibroblast-like Synoviocytes

【目的】探讨长链非编码RNA GAPLINC对类风湿关节炎成纤维样滑膜细胞(RA-FLS)增殖表型的影响。【方法】收集2022年1月至2022年12月于海南医学院附属海南医院行滑膜清理术或膝关节置换术的4例RA患者及3例创伤性关节炎废弃滑膜组织,分离培养并鉴定FLS细胞。通过qPCR法检测RA组和创伤组滑膜细胞内Ln⁃cRNA GAPLINC表达水平。利用siRNA干扰技术靶向敲低GAPLINC表达,通过EdU法检测各组细胞增殖水平、流式细胞术检测各组细胞的细胞周期分布情况。通过qPCR法与WB法检测细胞周期调控蛋白cyclin D1、PCNA及周期抑制蛋白P21的表达水平。【结果】本研究通过qRCR验证发现,RA组LncRNA GAPLINC表达水平明显高于创伤组(P<0.05);特异性siRNA对GAPLINC的干扰效率为(85.96±1.76)%(P<0.05)。敲低GAPLINC后,EdU细胞增殖实验显示,与空白对照组(6.02±0.48)%、溶剂对照组(6.32±1.48)%和阴性对照组(6.12±1.51)%相比,GAPLINC干扰组S期细胞占(2.89±1.53)%,较各对照组明显降低(P<0.05)。利用流式细胞术检测各组细胞周期分布,结果显示GAPLINC干扰组PI值,即S+G2/M期细胞所占的百分比为(14.92±5.34)%,明显低于空白对照组(29.68±8.48)%、溶剂对照组(28.97±6.82)%、阴性对照组(31.13±5.13)%(P<0.05)。敲低GAPLINC后,与增殖表型正相关的基因Cyclin D1和PCNA的mRNA和蛋白水平均下降(P<0.05),而与增殖表型负相关的基因p21表达上调(P<0.05)。【结论】Ln⁃cRNA GAPLINC在RA-FLS细胞中高表达,敲低GAPLINC表达后,能显著降低RA-FLS细胞增殖活性,抑制细胞周期转化,提示LncRNA GAPLINC可能影响RA-FLS细胞增殖表型,导致滑膜异常增生,参与RA疾病的进展。

【Objective】To investigate the effect of LncRNA GAPLINC on the cell proliferation of RA-FLSs.【Meth⁃ods】RA-FLSs were cultured from synovial specimens.The expression of LncRNA GAPLINC in RA-FLSs and trauma-FLSs groups was detected by qRCR.GAPLINC suppression was transfected by siRNA and the inhibition efficiency was de⁃tected by qRCR.Flow cytometry was adopted to determine the change of cell growth and cell cycle distribution.【ResμLts】The expression of LncRNA GAPLINC was significantly higher in RA-FLSs than that of the trauma-FLSs(P<0.05).Transfection of GAPLINC-siRNA significantly decreased the expression of LncRNA GAPLINC.GAPLINC silence in RA-FLSs revealed significant inhibition in cell proliferation which was showed by the reduced cell number in S phase(P<0.05).Moreover,flow cytometry assay showed GAPIINC-siRNA treatment group had an accumμLation of cells in the G0/G1 phase and decreased RA-FLSs in the S and G2/M phase(P<0.05).After GAPLINC knockdown,mRNA and protein levels of Cyclin D1 and PCNA,which were positively correlated with proliferative phenotype,were decreased(P<0.05),while p21,which was negatively correlated with proliferative phenotype,was up-regμLated(P<0.05).【Conclusions】The mRNA expression of GAPLINC was higher in RA-FLSs compared with trauma-FLSs,which was statistically significant(P<0.05).The silence of LncRNA GAPLINC coμLd significantly inhibit RA-FLSs cell growth and suppress the cell cycle transformation,which suggests that GAPLINC may play a role in the regμLation of proliferation of RA-FLSs,leading to sy⁃novial hyperplasia and contributing to RA progression.