miR-29b-3p介导HNF-4α抑制凝血因子Ⅹ表达在创伤性凝血病的机制研究  

Study on the mechanism of HNF-4αmediated by miR-29b-3p to inhibit the expression of coagulation factorⅩin trauma induced coagulopathy

作　　者：陈亮 贺慈 罗金花 黄志桃 CHEN Liang;HE Ci;LUO Jinhua;HUANG Zhitao(Trauma Center,The Third Affiliated Hospital of University of South China,Hengyang,Hunan 421900,China)

机构地区：[1]南华大学附属第三医院创伤中心,湖南衡阳421900

出　　处：《重庆医学》2025年第3期573-579,共7页Chongqing Medical Journal

基　　金：湖南省卫生健康委科研计划项目(202217015220)。

摘　　要：目的探讨微小RNA(miR)-29家族在创伤性凝血病(TIC)中的功能作用及具体机制。方法采用生物信息学分析miR-29家族成员与肝细胞核因子-4α(HNF-4α)的靶向关系。HE染色结果、血栓弹力图(TEG)参数与凝血4项指标验证TIC大鼠模型构建情况。采用逆转录实时荧光定量聚合酶链反应(RT-qPCR)及Western blot检测大鼠肝组织中miR-29家族、HNF-4α及凝血因子Ⅹ(FⅩ)表达情况;将过表达miR-29b-3p(miR-29b-3p mimics)或沉默miR-29b-3p(miR-29b-3p inhibitor)转染至肝细胞,RT-qPCR及Western blot检测肝细胞中miR-29b-3p、HNF-4α及FⅩ水平;双荧光酶报告基因实验验证miR-29b-3p对HNF-4α的靶向调控作用;将miR-29b-3p mimics和(或)HNF-4α过表达载体转染至肝细胞,RT-qPCR及Western blot检测肝细胞中miR-29b-3p、HNF-4α及FⅩ水平。结果miRDIP数据库预测结果显示,miR-29家族中的miR-29a-3p、miR-29b-3p、miR-29b-5p、miR-29c-3p均被发现与HNF-4α存在结合位点。HE染色结果、TEG参数与凝血4项指标表明TIC大鼠模型构建成功。RT-qPCR及Western blot检测显示,与对照组相比,模型组HNF-4α、FⅩ水平明显下调,miR-29b-3p在TIC大鼠中明显上调(P<0.01);细胞实验显示,miR-29b-3p mimics及miR-29b-3p inhibitor对肝细胞增殖无明显影响(P>0.05),miR-29b-3p mimics可明显下调HNF-4α、FⅩ表达,miR-29b-3p inhibitor则可明显上调HNF-4α、FⅩ表达水平(P<0.05)。双荧光素酶报告基因实验显示,HNF-4α与miR-29b-3p之间存在靶向结合。HNF-4α过表达则可抵消过表达miR-29b-3p对FⅩ的抑制作用(P<0.05)。结论miR-29b-3p在TIC大鼠中明显上调,可通过靶向HNF-4α介导调控FⅩ表达水平促进TIC进展。Objective To investigate the function and mechanism of miR-29 family in trauma induced coagulopathy(TIC).Methods Bioinformatics was used to analyze the targeting relationship between miR-29 family members and hepatocyte nuclear factor-4α(HNF-4α).HE staining results,TEG parameters and coagulation parameters were used to verify the TIC rat model construction.Real-time quantitative fluorescent PCR(RT-qPCR)and Western blot were used to detect the expression of miR-29 family,HNF-4αand coagulation factorⅩ(FⅩ)in rat liver tissues.Overexpression of miR-29b-3p(miR-29b-3p mimics)or silence of miR-29b-3p(miR-29b-3p inhibitor)was transfected into hepatocytes,and the levels of miR-29b-3p,HNF-4αand FⅩin hepatocytes were detected by RT-qPCR and Western blot.Double lucifase reporter gene assay verified the targeted regulation of miR-29b-3p on HNF-4α.The miR-29b-3p mimics and/or HNF-4αoverexpression vector were transfected into hepatocytes,and the levels of miR-29b-3p,HNF-4αand FⅩin hepatocytes were detected by RT-qPCR and Western blot.Results Bioinformatic prediction results from the miRDIP database identified that multiple members of the miR-29 family(miR-29a-3p,miR-29b-3p,miR-29b-5p,and miR-29c-3p)contain potential binding sites with HNF-4α.Histopathological evaluation through HE staining,combined with TEG parameters and coagulation profiles,confirmed successful establishment of the TIC rat model.Quantitative analyses using RT-qPCR and Western blot revealed that compared to controls,both HNF-4αand coagulation FⅩexpression levels were markedly suppressed in the model group,while miR-29b-3p expression showed significant elevation in TIC rats(P<0.01).In vitro functional studies demonstrated that neither overexpression nor silencing of miR-29b-3p significantly influenced hepatocyte proliferation(P>0.05).However,forced expression of miR-29b-3p effectively downregulated HNF-4αand its downstream target FⅩ,whereas miR-29b-3p knockdown substantially upregulated these molecules(P<0.05).This regulatory relationship

关 键 词：创伤性凝血病 微小RNA 肝细胞核因子4Α 凝血因子Ⅹ 生物信息学 

分 类 号：R641[医药卫生—外科学]