LOC152742通过TLR4信号通路和炎症反应调控结核分枝杆菌对Ⅱ型肺泡上皮细胞的感染

LOC152742 regulates Mycobacterium tuberculosis infection of typeⅡalveolar epithelial cells via TLR4 signaling pathway and inflammatory response

目的探讨长链非编码RNA(lncRNA)LOC152742对结核分枝杆菌感染的人Ⅱ型肺泡上皮细胞炎症反应和Toll样受体4(TLR4)信号通路的影响。方法在A549细胞中转染LOC152742 siRNA,实验分为对照(Con)组、感染(H37Rv)组、转染对照(H37Rv+si-NC)组和转染(H37Rv+si-LOC152742)组。RT-PCR分析LOC152742表达水平变化,MTT法分析A549细胞增殖率,流式细胞术检测细胞凋亡情况,蛋白免疫印迹(Western blot)法检测活化的半胱氨酸天冬氨酸蛋白酶-3(cleaved caspase-3)、活化的半胱氨酸天冬氨酸蛋白酶-9(cleaved caspase-9)、TLR4、髓样分化因子(MyD88)蛋白表达,ELISA分析细胞上清中白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)的含量。在H37Rv+si-LOC152742组A549细胞中添加TLR4特异性抑制剂TAK242,检测TAK242对炎症反应的影响。结果与Con组相比,H37Rv组A549细胞凋亡率、LOC152742、cleaved caspase-3、cleaved caspase-9、TLR4和MyD88的表达以及IL-6、IL-1β和TNF-α的含量显著升高(P<0.05),细胞增殖率显著降低(P<0.05);与H37Rv组相比,H37Rv+si-LOC152742组A549细胞凋亡率、LOC152742、cleaved caspase-3、cleaved caspase-9、TLR4和MyD88的表达以及IL-6、IL-1β和TNF-α的含量显著降低(P<0.05),细胞增殖率显著升高(P<0.05);TLR4特异性抑制剂TAK242能够逆转下调LOC152742对结核分枝杆菌感染的A549细胞的影响。结论H37Rv感染的A549细胞中LOC152742的表达上调,下调LOC152742能够通过抑制TLR4信号通路减轻炎症反应调控结核分枝杆菌对肺泡上皮细胞的感染。

This study was performed to investigate the effect of long-non-coding RNA(lncRNA)LOC152742on the inflammatory response and Toll-like receptor 4(TLR4)signaling pathway in human type II alveolar epithelial cells infected by Mycobacterium tuberculosis(Mtb).Human type II alveolar epithelial cells A549 were recruited and divided into control(Con)group,infection(infected with Mtb strain H37Rv)group,transfection control(H37Rv+siNC)group and transfection(H37Rv+si-LOC152742)group.The expression level of LOC152742 was analyzed by RT-PCR,the proliferation and apoptosis of A549 cells were analyzed by thiazolyl blue(MTT)and flow cytometry;Western blot method was used to detect the expression levels of activated caspase-3(cleaved caspase-3),activated caspase-9(cleaved caspase-9),TLR4,myeloid differentiation factor(MyD88)protein;ELISA was employed to analyze the levels of interleukin-6(IL-6),interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in cell supernatant.The TLR4-specific inhibitor TAK242 was added to the A549 cells of the H37Rv+si-LOC152742 group to detect the effect of TAK242 on the inflammatory response.Data showed that compared with the Con group,the apoptosis rate,the expression of LOC152742,cleaved caspase-3/9,TLR4 and MyD88,and the levels of IL-6,IL-1βand TNF-αwere significantly increased in the H37Rv group(P<0.05),while the cell proliferation rate was significantly decreased(P<0.05).As compared with the H37Rv group,the A549 cell apoptosis rate,LOC152742,cleaved caspase-3/9,TLR4,MyD88,and the levels of IL-6,IL-1βand TNF-αwere significantly decreased in the H37Rv+si-LOC152742 group(P<0.05),while the cell proliferation rate was significantly increased(P<0.05).The TLR4-specific inhibitor TAK242 was able to reverse the effects of LOC152742 down-regulation on Mtb-infected A549 cells.In conclusion,the expression of LOC152742 is up-regulated in H37Rv-infected A549 cells,and down-regulation of LOC152742 can reduce the inflammatory response and regulate Mtb infection of alveolar epithelial cells by inhibiting the TLR4 signaling pathway.