微小RNA-224通过靶向抑制p21参与调控脂多糖诱导的肺微血管内皮细胞损伤研究

MicroRNA-224 mediates lipopolysaccharide-induced injury of pulmonary microvascular endothelium cells via regulating p21

目的探讨miR-224在脂多糖(LPS)诱导的肺微血管内皮细胞(PMVEC)损伤中的作用及机制。方法分离清洁级BALB/c小鼠(购自浙江大学实验动物中心)原代PMVEC并体外培养,使用1.0 mg/L LPS处理PMVEC以诱导细胞损伤。通过转染miR-224抑制序列或p21的小干扰RNA(siRNA)序列分别下调PMVEC的微小RNA-224(miR-224)及p21表达水平。采用细胞计数试剂盒法及流式细胞仪检测PMVEC的细胞活力变化及凋亡率变化。实时荧光定量聚合酶链反应(FQ-PCR)及蛋白质印迹法(Western blot)检测PMVEC的miR-224及p21表达水平。使用双荧光素酶报告实验分析miR-224及p21的靶向关系。两组间的均数比较采用t检验,多组间的均数两两比较在采用单因素方差分析基础上使用最小显著差异法(LSD)检验。结果与未经任何处理的PMVEC比较,LPS处理后细胞相对细胞活力降低至(42.333±7.586)%,凋亡率提高至(32.141±2.449)%,miR-224表达增加至1.791±0.167,p21 mRNA水平降低至0.527±0.058,以上差异有统计学意义(t=8.532、7.261、7.113及8.467,P值均<0.01)。与单纯LPS处理的细胞比较,miR-224下调的细胞在LPS处理后的相对细胞活力增加且凋亡率显著降低,差异有统计学意义(F=62.618、32.643,P<0.01)。抑制p21表达会消除miR-224下调带来的这种保护作用。结论miR-224可能通过靶向抑制p21参与调控LPS诱导的PMVEC损伤。抑制miR-224可能有助于脓毒症后急性肺损伤或急性呼吸窘迫综合征的防治。

Objective To investigate the effects of microRNA(miRNA,miR)-224 level changes on the lipopolysaccharide(LPS)-induced injury of pulmonary microvascular endothelium cells(PMVECs)and its mechanism.Methods Primary PMVECs were isolated from clean-level BALB/c mice and cultured in vitro.In order to induce cells injury,1.0 mg/L LPS was used to treat PMVECs.MiR-224 inhibitor and p21 siRNA were transfected for silencing miR-224 and p21,respectively.The cell viability and apoptosis rate of PMVECs were detected by cell counting kit-8 reagent and flow cytometry,respectively.The changes of miR-224 and p21 were detected by fluorescent quantitative polymerase chain reaction(FQ-PCR)and Western blotting.Dual-luciferase reporter assay was used to determine the target relationship between miR-224 and p21.T-test was used to compare the mean between the two groups and the pairwise comparison of the mean among multiple groups was conducted by LSD test on the basis of one-way analysis of variance.Results After LPS treatment,the relative cell viability decreased to(42.333±7.586)%and apoptosis rate increased to(32.141±2.449)%as compared with non-LPS treated cells.Meanwhile,the level of miR-224 increased to 1.791±0.167 with a decreased p21 mRNA expression(0.527±0.058)(t=8.532,7.261,7.113 and 8.467,P<0.01).As compared with simple LPS treated cells,PMVECs transfected with miR-224 inhibitor showed lower cell inhibition and apoptosis rate after LPS treatment(F=62.618 and 32.643,P<0.01).However,p21 knock-down could antagonize the protective effect of miR-224.Conclusion MiR-224 may mediate the LPS-induced injury of PMVECs via targeting p21.MiR-224 could be a therapy target for acute lung injury/acute respiratory distress syndrome after sepsis.