沉默circANKRD11靶向miR-218-5p对RSV诱导的支气管上皮细胞损伤的影响

The effects of silencing circANKRD11 on RSV-induced bronchial epithelial cell damage by targeting miR-218-5p

目的探究沉默circANKRD11对呼吸道合胞病毒(RSV)诱导的支气管上皮细胞损伤的影响及其可能作用机制。方法采用RSV诱导人支气管上皮细胞HBECs建立细胞损伤模型,相应转染后用含有感染复数为0.0001 RSV的培养基培养24 h;qRT-PCR法检测circANKRD11、miR-218-5p、TNFR1和BRD4的表达量;CCK-8法、流式细胞术分别检测细胞增殖及凋亡;ELISA法检测IL-6、IL-1β的水平;双荧光素酶报告实验检测circANKRD11与miR-218-5p的靶向关系;Western blot法检测Bax、Bcl-2、TNFR1和BRD4蛋白表达量。结果RSV诱导的HBECs中circANKRD11的表达量升高(P<0.05),miR-218-5p的表达量降低(P<0.05),TNFR1和BRD4表达量升高(P<0.05);转染si-circANKRD11或转染miR-218-5p mimic后,RSV诱导的HBECs细胞增殖的抑制率下降,炎症因子含量降低,凋亡细胞数减少(P<0.05);circANKRD11可充当miR-218-5p的分子海绵而靶向结合miR-218-5p;共转染si-circANKRD11与miR-218-5p inhibitor可恢复转染si-circANKRD11对RSV诱导的HBECs增殖、凋亡及炎症的作用。结论沉默circANKRD11可通过上调miR-218-5p表达抑制TNFR1和BRD4表达,促进细胞增殖及抑制细胞凋亡和炎症反应从而减轻RSV诱导的支气管上皮细胞损伤。

This study was designed to explore the effect of silencing circANKRD11 on bronchial epithelial cell damage induced by respiratory syncytial virus(RSV)and its possible mechanism.Human bronchial epithelial cells(HBECs)induced by RSV were cultured for 24 h in medium with RSV of 0.0001 MOI after corresponding transfection to construct cell damage model.qRT-PCR method was used to detect the expression of circANKRD11,miR-218-5p,TNFR1 and BRD4;CCK-8 method and flow cytometry were used to detect cell proliferation and apoptosis;ELISA method was used to detect the levels of IL-6 and IL-1β.Furthermore,the dual luciferase reporter experiment was used to detect the targeting relationship between circANKRD11 and miR-218-5p;Western blot method was used to detect the expression of Bax,Bcl-2,TNFR1,and BRD4 protein.Data showed that the expression of circANKRD11,TNFR1 and BRD4 in RSV-induced HBECs was increased(P<0.05),while the expression of miR-218-5p was decreased(P<0.05).After transfection of si-circANKRD11 or miR-218-5p mimic,reductions in cell proliferation inhibition rate,IL-6 level,IL-1βlevel and apoptosis rate were observed in RSVinduced HBECs(P<0.05).circANKRD11 could act as a molecular sponge to targeting bind to miR-218-5p.The introduction of miR-218-5p inhibitor in cells was able to abolish the effects of si-circANKRD11 transfection on the proliferation,apoptosis and inflammation of RSV-induced HBECs.Taken together,silencing of circANKRD11 could promote cell proliferation and inhibit cell apoptosis and inflammation by decreasing TNFR1 and BRD4 level via elevating miR-218-5p level,thereby reducing RSV-induced bronchial epithelial cell damage.