摘要
目的 探讨长链非编码(long non-coding,Lnc)核糖核酸(ribonucleic acid,RNA)X-非活性特异性转录本(X-inactive specific transcript,XIST)调节微小RNA(microRNA,miR)-574-5p/Toll样受体(Toll-like receptor 4,TLR4)轴对呼吸道合胞病毒(respiratory syncytial virus,RSV)感染的支气管上皮细胞凋亡的影响.方法 将人支气管上皮细胞(human bronchial epithelial,16HBE)分为阴性对照(negative control,NC)组、RSV感染(RSV infection,RSV)组、小干扰RNA阴性对照(small interfering RNA negative control,si-NC)组、XIST小干扰RNA(XIST small interfering RNA,si-XIST)组、si-XIST+抑制剂阴性对照(inhibitor negative control,inhibitor-NC)组、si-XIST+miR-574-5p抑制剂(miR-574-5p inhibitor)组,实时荧光定量聚合酶链反应检测LncRNA XIST、miR-574-5p表达;5-乙炔基-2'脱氧尿嘧啶核苷(5-ethynyl-2'-deoxyuridine,EdU)染色、流式细胞术分别检测细胞增殖、凋亡;酶联免疫吸附试验检测肿瘤坏死因子(tumor necrosis factor,TNF)-α、白细胞介素(interleukin,IL)-6、IL-1β 的分泌;双荧光素酶报告基因实验验证miR-574-5p与LncRNA XIST和TLR4的关系;蛋白质印迹检测TLR4、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、细胞抗凋亡因子B淋巴细胞瘤-2(B lymphoblastoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)、切割型半胱氨酸天冬氨酸蛋白水解酶-3(cleaved cysteine aspartate proteolytic enzyme 3,Cleaved Caspase-3)蛋白表达.结果 相较于NC组,RSV组、si-NC组LncRNA XIST、凋亡率、IL-6、IL-1β、TNF-α 水平、TLR4、Bax、Cleaved Caspase-3表达升高,EdU阳性率、miR-574-5p、PCNA、Bcl-2表达降低(P均<0.05);相较于si-NC组,si-XIST组LncRNA XIST表达、凋亡率、IL-6、IL-1β、TNF-α水平、TLR4、Bax、Cleaved Caspase-3表达降低,EdU阳性率、miR-574-5p、PCNA、Bcl-2表达升高(P均<0.05);下调miR-574-5p可逆转敲低LncRNA XIST对RSV感染的16HBE细胞凋亡和炎症反应的抑制(P<0.05);双荧光素酶报告基因实验证实miR-574-5p与LncRNA XIST、miR-574-5p与TLR4存在靶向调控关系(P<0.05).结论 LncRNA XIST在RSV感染的16HBE细胞中上调表达,敲低LncRNA XIST可能通过上调miR-574-5p来下调TLR4表达,抑制RSV感染后的16HBE细胞凋亡.
Abstract
Objective To investigate the effect of long non-coding (Lnc) ribonucleic acid (RNA) X-inactive specific transcript (XIST) on apoptosis of bronchial epithelial cells infected with respiratory syncytial virus (RSV) by regulating the microRNA (miR)-574-5p/Toll-like receptor 4 (TLR4) axis. Methods Human bronchial epithelial cells (16HBE) were separated into negative control (NC) group,RSV infection (RSV) group,small interfering RNA negative control (si-NC) group,XIST small interfering RNA (si-XIST) group,si-XIST+inhibitor negative control (inhibitor NC) group,and si-XIST+miR-574-5p inhibitor group,Real-time fluorescence quantitative polymerase chain reaction was applied to detect the expression of LncRNA XIST and miR-574-5p;5-ethynyl-2'-deoxyuridine (EdU) staining and flow cytometry were applied to detect cell proliferation and apoptosis,respectively;enzyme linked immunosorbent assay was applied to detect the secretions of tumor necrosis factor (TNF)-α,interleukin (IL)-6,IL-1β;dual luciferase reporter gene experiment was applied to verify the relationship between miR-574-5p and LncRNA XIST,TLR4. Western blot was applied to detect the expression of TLR4,proliferating cell nuclear antigen (PCNA),anti apoptotic factor B cell lymphomatoma-2 (Bcl-2),Bcl-2 associated X protein (Bax),and cleaved cysteine aspartate proteolytic enzyme 3 (Cleaved caspase-3) proteins. Results Compared with the NC group,the level of LncRNA XIST,apoptosis rate,the level of IL-6,IL-1β,TNF-α,the expression of TLR4,Bax,and Cleaved Caspase-3 in RSV group and si-NC group were elevated,the positive rate of EdU,the level of miR-574-5p,the expression of PCNA and Bcl-2 were decreased (P<0.05);compared with the si-NC group,the level of LncRNA XIST,apoptosis rate,the level of IL-6,IL-1β,TNF-α,the expression of TLR4,Bax,and Cleaved Caspase-3 in the si-XIST group were decreased,the positive rate of EdU,the level of miR-574-5p,the expression of PCNA and Bcl-2 were increased (P<0.05);downregulation of miR-574-5p was able to reverse the inhibitory effects of knocking down LncRNA XIST on apoptosis and inflammatory response of 16HBE cells infected with RSV (P<0.05);dual luciferase reporter gene experiment confirmed the targeted regulatory relationship between miR-574-5p and LncRNA XIST,and between miR-574-5p and TLR4 (P<0.05). Conclusion LncRNA XIST is upregulated in 16HBE cells infected with RSV,and knocking down LncRNA XIST may down-regulate the expression of TLR4 by up-regulating miR-574-5p,and inhibit apoptosis of 16HBE cells after RSV infection.
基金项目
2023年市级科技计划自筹经费项目(2322087D)
维普
万方数据