lncRNA-AC013472.3对脂多糖刺激大鼠肺泡巨噬细胞分泌肿瘤坏死因子α的影响

Effect of lncRNA-AC013472.3 on LPS-stimulated secretion of tumor necrosis factor-αin NR8383 rat alveolar macrophages

目的探讨长链非编码RNA(lncRNA)-AC013472.3对脂多糖(LPS)刺激大鼠肺泡巨噬细胞(NR8383细胞)分泌肿瘤坏死因子(TNF)-α的影响。方法以NR8383细胞分别建立lncRNA-AC013472.3沉默及过表达模型,沉默模型分为乱序无意义阴性小干扰RNA序列(si-con)组(转染si-con的NR8383细胞)、LPS+si-con组(10μg/L的LPS处理转染si-con的NR8383细胞24 h)、小干扰RNA(siRNA)组(转染siRNA的NR8383细胞)和LPS+siRNA组(10μg/L的LPS处理转染siRNA的NR8383细胞24 h);过表达模型分为空质粒(p-con)组(转染p-con的NR8383细胞)、LPS+p-con组(10μg/L的LPS处理转染p-con的NR8383细胞24 h)、lncRNA过表达质粒(plncRNA)组(转染plncRNA的NR8383细胞)和LPS+plncRNA组(10μg/L的LPS处理转染plncRNA的NR8383细胞24 h),实时荧光定量PCR(qPCR)方法检测各组细胞中TNF-αmRNA相对表达量;Western印迹法检测TNF受体相关因子-6(TRAF-6)和磷酸化的核因子-κB(NF-κB)p65蛋白相对表达量。结果在沉默模型中,LPS+si-con组TNF-αmRNA相对表达量、TRAF-6和NF-κB p65蛋白相对表达量均显著高于si-con组(2.040±0.195比1.048±0.207、0.473±0.022比0.293±0.076和0.469±0.062比0.252±0.038)(均P<0.05);LPS+siRNA组TNF-αmRNA相对表达量、TRAF-6和NF-κB p65蛋白相对表达量均显著高于siRNA组(4.158±0.119比1.028±0.019、0.700±0.104比0.231±0.023和0.771±0.095比0.258±0.050)(均P<0.05);LPS+siRNA组各指标相对表达量显著高于LPS+si-con组(均P<0.05)。在过表达模型中,LPS+p-con组TNF-αmRNA相对表达量、TRAF-6和NF-κB p65蛋白相对表达量均显著高于p-con组(1.961±0.169比0.999±0.143、0.533±0.047比0.247±0.020和0.565±0.108比0.276±0.048)(均P<0.05);LPS+plncRNA组TNF-αmRNA、TRAF-6和NF-κB p65蛋白相对表达量均显著高于plncRNA组(1.322±0.110比1.043±0.093、0.347±0.035比0.232±0.023和0.405±0.072比0.268±0.031)(均P<0.05);LPS+plncRNA组各指标相对表达量显著低于LPS+p-con组(均P<0.05)。结论lncRNA-AC013472.3可能通过抑制NF-κB信号通路的激活,从而抑制LPS刺激NR8383细胞分泌TNF-α。

Objective To investigate the effect of long non-coding RNA-AC013472.3 on lipopolysaccharide(LPS)-stimulated secretion of tumor necrosis factor(TNF)-αin NR8383 rat alveolar macrophages.Methods Silencing and overexpression models of lncRNA-AC013472.3 were established with NR8383 rat alveolar macrophages as the experimental subjects.The silencing models were divided into three groups:random nonsense negative small interfering RNA sequence(si-con)group(si-con group,si-con transfected NR8383 cells),LPS+si-con group(10μg/L LPS was used to treat si-con transfected NR8383 cells for 24 h),and siRNA group(siRNA transfected NR8383 cells),and LPS+siRNA group(10μg/L LPS was used to treat siRNA transfected NR8383 cells for 24 h).The overexpression models were divided into the empty plasmid(p-con)group(p-con transfected NR8383 cells),LPS+p-con group(10μg/L LPS was used to treat p-con transfected NR8383 cells for 24 h),lncRNA overexpression plasmid(plncRNA)group(plncRNA transfected NR8383 cells),and the LPS+plncRNA group(10μg/L LPS was used to treat plncRNA transfected NR8383 cells for 24 h).The mRNA levels of TNF-αin each group were examined by quantitative real-time PCR(qPCR).The protein levels of tumor necrosis factor receptor-related factor-6(TRAF-6)and phosphorylated nuclear factor-κB(NF-κB)p65 were examined by Western blot.Results In the silencing model,the mRNA levels of TNF-α,the protein levels of TRAF-6 and NF-κB p65 in the LPS+si-con group were significantly higher than those in the si-con group(2.040±0.195 vs 1.048±0.207,0.473±0.022 vs 0.293±0.076 and 0.469±0.062 vs 0.252±0.038)(all P<0.05).The mRNA levels of TNF-α,the protein levels of TRAF-6 and NF-κB p65 in the LPS+siRNA group were significantly higher than those in the siRNA group(4.158±0.119 vs 1.028±0.019,0.700±0.104 vs 0.231±0.023 and 0.771±0.095 vs 0.258±0.050)(all P<0.05).The relative expression levels of all indexes in the LPS+siRNA group were significantly higher than those in the LPS+si-con group(all P<0.05).In the overexpression model,the mRNA levels of TNF-α,the protein levels of TRAF-6 and NF-κB p65 in the LPS+p-con group were significantly higher than those in the p-con group(1.961±0.169 vs 0.999±0.143,0.533±0.047 vs 0.247±0.020 and 0.565±0.108 vs 0.276±0.048)(all P<0.05).The mRNA levels of TNF-α,the protein levels of TRAF-6 and NF-κB p65 in the LPS+plncRNA group were significantly higher than those in the plncRNA group(1.322±0.110 vs 1.043±0.093,0.347±0.035 vs 0.232±0.023 and 0.405±0.072 vs 0.268±0.031)(all P<0.05).The relative expression of all indexes in the LPS+plncRNA group were significantly lower than that in the LPS+p-con group(all P<0.05).Conclusion LncRNA-AC013472.3 may inhibit the activation of NF-κB signaling pathway,thereby inhibiting the LPS-stimulated secretion of TNF-αin NR8383 rat alveolar macrophages.