结直肠肿瘤差别表达基因对氧化型低密度脂蛋白诱导的巨噬细胞凋亡的影响及机制

Effect of lncRNA CRNDE on oxLDL-induced apoptosis of macrophages and its mechanism

目的探讨长链非编码RNA(lncRNA)结直肠肿瘤差别表达基因(CRNDE)对氧化型低密度脂蛋白(oxLDL)诱导的巨噬细胞凋亡和炎性因子分泌的影响及其机制。方法以0、25、50、100μmol/ml oxLDL处理小鼠巨噬细胞RAW264.724 h后,采用实时荧光定量PCR检测CRNDE表达。将RAW264.7巨噬细胞分为对照组(正常培养,n=3)、oxLDL组(以100μmol/ml oxLDL处理,n=3)、oxLDL+siNC组(转染阴性对照RNA后给予100μmol/ml oxLDL处理,n=3)和oxLDL+siCRNDE组(转染靶向CRNDE的小干扰RNA后给予100μmol/ml oxLDL处理,n=3),采用流式细胞仪检测细胞凋亡率,免疫印迹法检测B淋巴细胞瘤2基因(Bcl-2)、Bax、磷酸化NF-κB(p-NF-κB)P65、NF-κB抑制蛋白(IκBa)和磷酸化IκBa(p-IκBa)蛋白表达。结果0、25、50、100μg/ml oxLDL刺激后巨噬细胞中lncRNA CRNDE表达水平比较,有统计学差异(1.00±0.07 vs 1.52±0.23 vs 2.28±0.59 vs 3.56±0.78,P<0.01)。与对照组比较,oxLDL组、oxLDL+siNC组和oxLDL+siCRNDE组巨噬细胞中CRNDE表达、细胞凋亡率、白细胞介素6(IL-6)、TNF-α、Bax、p-NF-κB P65、p-IκBa蛋白表达明显升高,Bcl-2蛋白表达明显降低(P<0.05);oxLDL+siCRNDE组巨噬细胞中lncRNA CRNDE表达、细胞凋亡率、IL-6、TNF-α、Bax、p-NF-κB P65及p-IκBa蛋白表达明显低于oxLDL组,Bcl-2蛋白表达明显高于oxLDL组(P<0.05)。结论下调CRNDE表达可抑制oxLDL诱导的巨噬细胞凋亡和炎性因子分泌,其作用机制可能与抑制NF-κB信号通路的活化有关。

Objective To study the effect of lncRNA CRNDE gene on oxLDL-induced apoptosis of macrophages and secretion of inflammatory factors and its mechanism.Methods The expression of CRNDE gene was detected by RT-fluorescent qPCR after the macrophages were treated in 0μmol/ml oxLDL,25μmol/ml oxLDL,50μmol/ml oxLDL and 100μmol/ml oxLDL respectively for 24 h.The in vitro cultured macrophages were divided into control group(treated in normal culture,n=3),oxLDL group(treated in 100μmol/ml oxLDL,n=3),oxLDL+siNC group(transfected with NC-siRNA and treated in 100μmol/ml oxLDL,n=3)and oxLDL+siCRNDE gene group(treated in 100μmol/ml oxLDL after transfection with CRNDE-siRNA,n=3).The expression of CRNDE gene was detected by RT-fluorescent qPCR and flow cytometry respectively.The apoptosis of macrophages was assayed by flow cytometry.The expressions of Bcl-2,Bax,NF-κB P65,IκBa,p-NF-κB P65 and p-IκBa were detected by Western blot.Results The expression level of lncRNA CRNDE gene was significantly lower in macrophages treated in 0μg/ml oxLDL than in those treated in 25μmol/ml oxLDL,50μmol/ml oxLDL and 100μmol/ml oxLDL(1.00±0.07 vs 1.52±0.23 vs 2.28±0.59 vs 3.56±0.78,P<0.01).The apoptosis rate of macrophages and the expression levels of CRNDE gene,IL-6,TNF-α,Bax,p-NF-κB P65,p-IκBa were significantly higher while the expression level of Bcl-2 was significantly lower in oxLDL group,oxLDL+siNC group and oxLDL+siCRNDE group than in control group(P<0.05).The apoptosis rate of macrophages and the expression levels of CRNDE gene,IL-6,TNF-α,Bax,p-NF-κB P65,p-IκBa were significantly lower while the expression level of Bcl-2 was significantly higher in oxLDL+siCRNDE group than in oxLDL group(P<0.05).Conclusion Down regulating the expression of CRNDE gene suppresses oxLDL-induced apoptosis of macrophages and secretion of inflammatory factors by inhibiting the NF-κB signaling pathway activation.