LncRNA PCED1B-AS1调控NLRP3促进巨噬细胞清除结核分枝杆菌的机制研究

Study on mechanism of lncRNA PCED1B-AS1 regulating NLRP3 to promote clearance of Mycobacterium tuberculosis by macrophages

目的研究长链非编码RNA PCED1B-AS1调控核苷酸结合寡聚化结构域样受体3(NLR family pyrindomain containing 3,NLRP3)对巨噬细胞清除结核分枝杆菌和分泌炎症因子的影响。方法在巨噬细胞RAW264.7中转染pcDNAPCED1B-AS1,并用结核分枝杆菌感染,qRT-PCR方法检测PCED1B-AS1和TNF-α、IL-6 mRNA水平,用菌落形成实验检测巨噬细胞对结核分枝杆菌的清除作用,Western blot方法检测细胞中NLRP3蛋白表达水平。将NLRP3小干扰RNA(small interfering RNA,siRNA)和pcDNA-PCED1B-AS1共转染到巨噬细胞中,用结核分枝杆菌感染,同样检测细胞中TNF-α、IL-6 mRNA水平和菌落量。结果结核分枝杆菌感染后的巨噬细胞中PCED1B-AS1水平降低,TNF-α、IL-6 mRNA水平升高。转染pcDNA-PCED1B-AS1后的巨噬细胞经过结核分枝杆菌感染以后,细胞中PCED1B-AS1、TNF-α、IL-6 mRNA水平升高,形成的菌落量减少,细胞中NLRP3蛋白表达水平升高。NLRP3 siRNA可以逆转过表达PCED1B-AS1对结核分枝杆菌感染的巨噬细胞中TNF-α、IL-6 mRNA表达和菌落量形成的影响。结论上调PCED1B-AS1促进巨噬细胞清除结核分枝杆菌,诱导细胞表达TNF-α、IL-6 mRNA的机制与上调NLRP3表达有关。

Objective To study the effect of long-non-coding RNA(lncRNA)PCED1 B-AS1 regulating NLR family pyrin domain containing 3(NLRP3)on the clearance of Mycobacterium tuberculosis and secretion of inflammatory factors by of levels of PCED1 B-AS1,TNF-αand IL-6 mRNA were detected by qRT-PCR method,and the elimination of Mycobacterium tuberculosis was detected by colony formation assay,the expression level of NLRP3 protein in cells was detected by Western blot.NLRP3 siRNA and pcDNA-PCED1 B-AS1 were co-transfected into macrophages and infected with Mycobacterium tuberculosis.The levels of TNF-α,IL-6 mRNA and the amount of colonies in the cells were also detected.Results The levels of PCED1 B-AS1 in macrophages after infection with Mycobacterium tuberculosis decreased,and the levels of TNF-αand IL-6 mRNA increased.After the macrophages transfected with pcDNA-PCED1 B-AS1 were infected with Mycobacterium tuberculosis,the levels of PCED1 B-AS1 in the cells increased,the levels of TNF-αand IL-6 mRNA increased,the number of colonies formed decreased,and the NLRP3 protein expression level in the cells increased.NLRP3 siRNA could reverse the effect of overexpression of PCED1 B-AS1 on the expression of TNF-α,IL-6 mRNA and the amount of colony formation in macrophages infected by Mycobacterium tuberculosis.Conclusions Up-regulation of PCED1 B-AS1 promotes macrophage clearance of Mycobacterium tuberculosis and induces the expression of TNF-αand IL-6 mRNA.The mechanism is related to the up-regulation of NLRP3 expression.