miR-28-5p靶向Nrf2调控LPS诱导的急性肺损伤机制研究

Mechanism of miR-28-5p Targeting Nrf2 to Regulate LPS-Induced Acute Lung Injury

目的探究miR-28-5p在脂多糖(LPS)诱导的急性肺损伤(ALI)进程中的作用和分子机制。方法将40只C57BL/6雄鼠按随机数字表法分为生理盐水组、ALI-3h组、ALI-6h组和ALI-12h组,每组10只。利用LPS诱导小鼠ALI 3h、6h、12h,取肺组织进行荧光定量PCR(RT-PCR)检测miR-28-5p表达;同时体外利用LPS刺激小鼠巨噬细胞RAW264.7发生炎症反应,RT-PCR检测细胞miR-28-5p表达;巨噬细胞过表达miR-28-5p mimic后利用LPS刺激,RT-PCR检测巨噬细胞炎症因子肿瘤坏死因子-α(TNF-α)、白细胞介素1β(IL-1β)表达情况;RT-PCR和Western blot检测抗氧化关键分子核因子E2相关因子2(Nrf2)、血红素加氧酶(HO-1)、醌氧化还原酶(NQO1)表达水平;Targetscan 7.0预测miR-28-5p靶向基因,突变结合位点后,并通过荧光素酶报告基因系统检测miR-28-5p对靶基因的转录调控作用。结果与生理盐水组比较,LPS诱导小鼠ALI 3h、6h、12h TNF-α、IL-1β水平明显升高[TNF-α:(6.24±1.84)、(4.31±0.97)、(2.97±0.49)比(1.00±0.21),P<0.05;IL-1β:(6.29±1.06)、(4.95±0.93)、(3.59±1.05)比(1.00±0.53),P<0.05],肺组织miR-28-5p水平明显升高[(7.24±1.35)、(5.26±1.64)、(3.24±0.97)比(1.00±0.34),P<0.05];与生理盐水组比较,LPS刺激巨噬细胞炎症反应3h、6h、12h细胞miR-28-5p水平明显升高[(5.21±0.15)、(3.51±0.78)、(2.45±0.41)比(1.00±0.35),P<0.05];与mimic-NC组比较,巨噬细胞过表达miR-28-5p mimic组细胞炎症因子TNF-α和IL-1β表达水平明显升高[TNF-α:(4.86±0.59)比(1.00±0.52);IL-1β:(3.54±0.41)比(1.00±0.24),P<0.05];同时与mimic-NC组比较,miR-28-5p mimic过表达组抗氧化关键分子Nrf2及其下游HO-1、NQO1表达明显升高[Nrf2:(5.16±1.03)比(1.00±0.14);HO-1:(4.91±1.08)比(1.00±0.12);NQO1:(5.61±0.80)比(1.00±0.08),P<0.05];Targetscan 7.0预测发现miR-28-5p靶向Nrf2基因3'UTR序列,荧光素酶报告基因实验进一步明确miR-28-5p通过靶向Nrf2基因3'UTR序列调控其转录。结论miR-28-5p在LPS诱导的ALI中表达上调,并进一步加剧ALI病理进程,其作用机制可能是通过靶向巨噬细胞Nrf2,从而减弱胞内抗氧化能力,促进炎症因子分泌和巨噬细胞的活化。

Objective To explore the role and molecular mechanism of miR-28-5p in the process of lipopolysaccharide(LPS)-induced acute lung injury(ALI).Methods Forty C57BL/6 male mice were randomly divided into normal saline group,ALI-3h group,ALI-6h group and ALI-12h group,with 10 animals in each group.LPS was used to induce mouse ALI for 3h,6h and 12h,and lung tissues were taken for fluorescence quantitative PCR(RT-PCR)to detect the expression of miR-28-5p.At the same time,LPS was used to stimulate the inflammatory reaction of mouse macrophage RAW264.7 in vitro,and the expression of miR-28-5p was detected by RT-PCR.Macrophages overexpressed miR-28-5p for mimic,then stimulated by LPS,and the expressions of inflammatory factors such as tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)in macrophages were tested by RT-PCR.RT-PCR and Western blot were used to detect the expression levels of nuclear factor E2 related factor 2(Nrf2),heme oxygenase(HO-1)and quinone oxidoreductase(NQO1).Targetscan 7.0 predicted the target gene of miR-28-5p,mutated the binding site,and detected the transcriptional regulation of miR-28-5p on the target gene by luciferase reporter system.Results Compared to normal saline group,TNF-αand IL-1βlevels of LPS induced-ALI 3h,6h and 12h group were significantly increased,i.e.,TNF-α:[(6.24±1.84),(4.31±0.97)and(2.97±0.49)vs(1.00±0.21)],IL-1β:[(6.29±1.06),(4.95±0.93)and(3.59±1.05)vs(1.00±0.53)],P<0.05,respectively;so was the level of miR-28-5p[(7.24±1.35),(5.26±1.64)and(3.24±0.97)vs(1.00±0.34),P<0.05].Compared to normal saline group,level of miR-28-5p in LPS-stimulated macrophage inflammatory response increased significantly at 3h,6h and 12h[(5.21±0.15),(3.51±0.78)and(2.45±0.41)vs(1.00±0.35),P<0.05].The expression levels of TNF-αand IL-1βin miR-28-5p mimic group were significantly higher than those in mimic NC group[TNF-α:(4.86±0.59)vs(1.00±0.52);IL-1β:(3.54±0.41)vs(1.00±0.24),P<0.05].Compared to mimic NC group,the expression of Nrf2,HO-1 and NQO1,the key antioxidant components in miR-28-5p mimic overexpression group,increased significantly[Nrf2:(5.16±1.03)vs(1.00±0.14);HO-1:(4.91±1.08)vs(1.00±0.12);NQO1:(5.61±0.80)vs(1.00±0.08),P<0.05].Targetscan 7.0 predicted that miR-28-5p targeted 3'-UTR of Nrf2 gene,and luciferase reporter gene experiment confirmed that miR-28-5p regulated Nrf2 gene transcription by targeting 3'-UTR of Nrf2.Conclusion The expression of miR-28-5p is up-regulated in LPS-induced ALI,which further aggravates the pathological process of ALI.The mechanism may be that miR-28-5p targets Nrf2 gene of macrophage,thereby weakening the intracellular antioxidant capacity and promoting the secretion of inflammatory factors and the activation of macrophages.