miR-133a-5p通过靶向细胞间黏附分子1对脂多糖诱导的肺泡上皮细胞A549损伤的影响

Effect of intercellular adhesion molecule-1-targeted miR-133a-5p on lipopolysaccharide-induced alveolar epithelial A549 cell injury

目的探讨miR-133a-5p靶向细胞间黏附分子1(ICAM1)对脂多糖(LPS)诱导的肺泡上皮细胞A549损伤的影响。方法双荧光素酶报告基因分析法验证miR-133a-5p对ICAM1的靶向作用。体外用LPS诱导A549细胞,分为对照组、LPS组、LPS+阴性对照(miR-NC)组、LPS+miR-133a-5p组、LPS+小干扰RNA(si)-NC组、LPS+si-ICAM1组。采用实时荧光定量PCR检测miR-133a-5p和ICAM1 mRNA的表达水平;流式细胞仪检测细胞凋亡;Western blot检测ICAM1、Bcl-2、Bax和活化半胱氨酸的天冬氨酸蛋白水解酶3(cleaved caspase-3)蛋白的表达;酶联免疫吸附检测白细胞介素6(IL-6)和肿瘤坏死因子α(TNF-α)的表达。结果与对照组比较,LPS组A549细胞miR-133a-5p的表达水平(0.39±0.04比1.00±0.09)显著降低,ICAM1的表达水平(0.86±0.08比0.39±0.03)显著升高,细胞凋亡率[(27.65±2.47)%比(8.13±0.89)%]显著升高,IL-6[(624.59±51.42)ng/L比(194.25±18.43)ng/L]和TNF-α[(548.35±51.42)ng/L比(174.26±19.43)ng/L]的分泌显著增加,差异均有统计学意义(均P<0.05)。与LPS+miR-NC组比较,LPS+miR-133a-5p组A549细胞凋亡率[(13.46±1.38)%比(28.71±2.54)%]显著降低,IL-6[(296.43±23.51)ng/L比(635.86±55.41)ng/L]和TNF-α[(321.14±30.56)ng/L比(563.24±49.52)ng/L]的分泌显著减少,差异均有统计学意义(均P<0.05);与LPS+si-NC组比较,LPS+si-ICAM1组A549细胞凋亡率[(13.65±1.64)%比(23.51±2.33)%]显著降低,IL-6[(324.15±29.41)ng/L比(625.39±52.59)ng/L]和TNF-α[(334.65±20.46)ng/L比(534.97±51.42)ng/L]的分泌显著减少,差异均有统计学意义(均P<0.05)。结论miR-133a-5p能减轻LPS诱导的肺泡上皮细胞损伤,其机制可能与下调ICAM1表达有关。

Objective To investigate the effect of intercellular adhesion molecule-1(ICAM1)targeted miR-133a-5p on lipopolysaccharide(LPS)-induced alveolar epithelial A549 cell injury.Methods Dual lucife-rase reporter assay was used to verify the ICAM1 targeted effect of miR-133a-5p.A549 cells were induced by LPS in vitro and divided into the control group,LPS group,LPS+negative control(miR-NC)group,LPS+miR-133a-5p group,LPS+small interfering RNA(si)-NC group,and LPS+si-ICAM1 group.The expression levels of miR-133a-5p and ICAM1 mRNA were detected by real-time fluorescent quantitative PCR.Apoptosis was detected by flow cytometry.The expression levels of ICAM1,Bcl-2,Bax and cleaved caspase-3 protein that could activate cysteine were detected by Western blot.The expression levels of interleukin-6(IL-6)and tumor necrosis factorα(TNF-α)were detected by enzyme linked immunosorbent assay kit.Results Compared with the control group,the LPS group had a decreased expression level of miR-133a-5p(0.39±0.04 vs.1.00±0.09)in A549 cells,increased expression of ICAM1(0.86±0.08 vs.0.39±0.03),an increased apoptotic rate[(27.65±2.47)%vs.(8.13±0.89)%],and increased secretion of IL-6[(624.59±51.42)ng/L vs.(194.25±18.43)ng/L]and TNF-α[(548.35±51.42)ng/L vs.(174.26±19.43)ng/L].The differences were significant(all P<0.05).Compared with the LPS+miR-NC group,the apoptosis rate of A549 cells[(13.46±1.38)%vs.(28.71±2.54)%]in LPS+miR-133a-5p group were significantly decreased,and the secretion of IL-6[(296.43±23.51)ng/L vs.(635.86±55.41)ng/L]and TNF-α[(321.14±30.56)ng/L vs.(563.24±49.52)ng/L]was significantly decreased(all P<0.05).Compared with LPS+si-NC group,the apoptosis rate of A549 cells[(13.65±1.64)%vs.(23.51±2.33)%]in LPS+si-ICAM1 group was significantly decreased,and the secretion of IL-6[(324.15±29.41)ng/L vs.(625.39±52.59)ng/L]and TNF-α[(334.65±20.46)ng/L vs.(534.97±51.42)ng/L]were significantly decreased(all P<0.05).Conclusions miR-133a-5p can alleviate LPS-induced alveolar epithelial cell injury,and the mechanism may be related to down-regulation of ICAM1 expression.