miR-21-5p通过靶向TGF-β1改善LPS诱导的脓毒症实验研究  

miR-21-5p Improving LPS-Induced Sepsis by Targeting TGF-β1

作　　者：雷磊[1] 赵鹏[1] 杨昌伟 刘传明 尹文[1] 李俊杰[1] LEI Lei;ZHAO Peng;YANG Chang-wei;LIU Chuan-ming;YIN Wen;LI Jun-jie(Department of Emergency Medicine,Xijing Hospital,Military Medical University of PLA Air Force,Xi'an 710032,China)

机构地区：[1]中国人民解放军空军军医大学西京医院急诊科,西安710032

出　　处：《解放军医药杂志》2020年第7期5-10,共6页Medical & Pharmaceutical Journal of Chinese People’s Liberation Army

基　　金：陕西省自然科学基金项目(2018KW-061)。

摘　　要：目的确定miR-21-5p与转化生长因子-β1(TGF-β1)的靶向关系,并探索miR-21-5p和TGF-β1相互作用对脂多糖(LPS)诱导的脓毒症小鼠的影响。方法构建LPS诱导的脓毒症小鼠模型,检测其降钙素和超敏C反应蛋白的水平以确定构建成功。将小鼠分为对照组、脓毒症组、NC antagomir组、miR-21-5p antagomir组、siRNA-NC组、si-TGF-β1组,每组6只。miR-21-5p antagomir组静脉注射miR-21-5p antagomir以低表达miR-21-5p,si-TGF-β1组静脉注射si-TGF-β1以低表达TGF-β1,通过qRT-PCR检测miR-21-5p和TGF-β1的表达情况。用Target Scan软件预测miR-21-5p与TGF-β1的靶位点,将miR-21-5p antagomir和si-TGF-β1分别转染293T细胞,用Western blot检测TGF-β1的表达水平,通过双荧光素酶报告分析测定野生型(TGF-β1-wt)和突变型(TGF-β1-mut)对荧光素酶活性影响,进一步确定miR-21-5p与TGF-β1的靶向关系。测定对照组、脓毒症组、miR-21-5p antagomir组、si-TGF-β1组小鼠血清中白介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、超氧化物歧化酶(SOD)、丙二醛(MDA)、降钙素和超敏C反应蛋白的水平。结果构建LPS诱导的脓毒症小鼠血清降钙素和超敏C反应蛋白、TGF-β1水平高于对照组,miR-21-5p低于对照组(P<0.05,P<0.01)。分别转染miR-21-5p antagomir和si-TGF-β1到模型小鼠后,miR-21-5p和TGF-β1表达显著降低,瞬转成功。在TGF-β1的3’UTR区425-432 bp区域有2个hsa-miR-21-5p潜在的靶向结合位点。与对照组比较,将si-TGF-β1转染到293T细胞后TGF-β1的表达量显著下降,而与对照组和转染si-TGF-β1组比较,转染miR-21-5p antagomir后TGF-β1的表达量显著提升(P<0.05,P<0.01)。相对于TGF-β1-mut,TGF-β1-wt能使荧光素酶的活性显著下降(P<0.05)。与对照组比较,脓毒症组小鼠血清IL-6、TNF-α、MDA、降钙素和超敏C反应蛋白的水平都显著提升,SOD活性显著下降(P<0.05,P<0.01);与脓毒症组比较,miR-21-5p antagomir组上述指标更加恶化,而si-TGF-β1组则显著改善Objective To determine the targeting relationship between miR-21-5p and transforming growth factor-β1(TGF-β1),and to investigate the effects of miR-21-5p and TGF-β1 interaction on LPS-induced sepsis in mice.Methods A mouse model of sepsis induced by LPS was constructed and the levels of calcitonin and hypersensitive C-reactive protein(hs-CRP)were measured to determine the success of the construction.The mice were divided into control group,sepsis group,NC antagomir group,miR-21-5p antagomir group,siRNA-NC group and si-TGF-β1 group(n=6 mice in each group).miR-21-5p antagomir group received intravenous injection of miR-21-5p antagomir and miR-21-5p was expressed at a low level,while si-TGF-β1 group received intravenous injection of si-TGF-β1 and TGF-β1 was expressed at a low level.The expression of miR-21-5p and TGF-β1 was detected by qRT-PCR.TargetScan was used to predict the target sites of miR-21-5p and TGF-β1,miR-21-5p antagomir and si-TGF-β1 were transfected into 293T cells,and the expression level of TGF-β1 was detected by Western blot.The effect of wild-type TGF-β1-(TGF-β1-wt)and mutant-type TGF-β1-(TGF-β1-mut)on luciferase activity was determined by dual luciferase reporter assay to further determine the targeting relationship between miR-21-5p and TGF-β1.Interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),superoxide dismutase(SOD),malondialdehyde(MDA),calcitonin and hs-CRP levels in the serum of mice in the control group,sepsis group,miR-21-5p antagomir group,and si-TGF-β1 group were determined.Results The levels of calcitonin,hs-CRP and TGF-β1 in mice with LPS-induced sepsis were higher than those in the control group,and miR-21-5p was lower than that in the control group(P<0.05,P<0.01).After transfection of miR-21-5p antagomir and si-TGF-β1 into model mice,the expression of miR-21-5p and TGF-β1 was significantly decreased,indicating that the transfection was successful.There were two potential targeting sites for hsa-miR-21-5p in the 425-432 bp region of the 3'UTR region of TGF-β1.C

关 键 词：脓毒症 miR-21-5p 转化生长因子β1 降钙素 白介素-6 肿瘤坏死因子-α 丙二醛 超氧化物歧化酶 小鼠 C57BL 

分 类 号：R-332[医药卫生] R631