miR-664b-3p调控Caspase-1介导细胞焦亡在深静脉血栓形成中的作用  

Regulatory role of miR-664b-3p in caspase-1-mediated pyroptosis in patients with deep vein thrombosis

作　　者：褚楚 刘文 郭红 赵霖 魏然 张振 郭强 朱肖肖 王彬[3] 李霞 CHU Chu;LIU Wen;GUO Hong;ZHAO Lin;WEI Ran;ZHANG Zhen;GUO Qiang;ZHU Xiaoxiao;WANG Bin;LI Xia(School of Basic Medicine(Institute of Basic Medicine),Shandong First Medical University,Jinan 250062,China;不详)

机构地区：[1]山东第一医科大学基础医学院(基础医学研究所),济南250062 [2]山东德升生物工程有限公司 [3]山东中医药大学附属医院

出　　处：《山东医药》2021年第8期20-23,共4页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81673981);山东省重点研发项目(2017GSF19118)。

摘　　要：目的观察深静脉血栓形成(DVT)患者外周血微小RNA-664b-3p(miR-664b-3p)、半胱氨酸天冬氨酸蛋白酶1(Caspase-1)水平变化,分析二者在细胞焦亡中的调控关系。方法选择健康志愿者(健康对照组)和DVT患者(DVT组)各30例,空腹抽取外周静脉血,分离血清及单个核细胞(PBMC)。Western blotting法检测PBMC中活化的半胱氨酸天冬氨酸蛋白酶1(Cleaved Caspase-1)、焦孔素D(GSDMD)活性片段(GSDMD-N)蛋白,qPCR法检测PBMC中的miR-664b-3p,ELISA法检测血清中的细胞焦亡相关炎症因子白细胞介素(IL)1β和IL-18蛋白。采用Target Scan 7.2生物信息学软件,预测miR-664b-3p与Caspase-1 mRNA的3′非翻译区(3′UTR)是否存在互补结合位点;分别构建包含结合位点的野生型及突变型Caspase-1 mRNA 3′UTR质粒,双荧光素酶报告基因系统检测miR-664b-3p对其荧光素酶报告基因活性的影响。结果DVT组PBMC中Cleaved Caspase-1、GSDMD-N蛋白表达及血清IL-1β、IL-18蛋白水平均高于健康对照组,miR-664b-3p表达低于健康对照组(P均<0.05);生物信息学分析显示,miR-664b-3p与Caspase-1 mRNA的3′UTR存在互补结合位点;转染miR-664b-3p mimics可以降低野生型Caspase-1 mRNA 3′UTR荧光素酶报告基因活性(P<0.05),但对突变型Caspase-1 mRNA 3′UTR报告基因活性无显著影响(P>0.05)。结论DVT患者PBMC中miR-664b-3p表达降低,可解除对靶基因Caspase-1的表达抑制,Caspase-1活性增强使GS⁃DMD-N表达升高,进而导致细胞焦亡及相关炎症因子(IL-1β、IL-18)表达升高,最终导致DVT。Objective To observe the changes of microRNA-664b-3p(miR-664b-3p)and Caspase-1 levels in the peripheral blood of patients with deep vein thrombosis(DVT),and to explore the regulatory relationship between them in pyroptosis.Methods Thirty cases of healthy subjects(healthy control group)and 30 cases of DVT patients(DVT group)were collected,and their peripheral blood on a empty stomach was drawn to separate serum and peripheral blood mononuclear cells(PBMCs),respectively.The activity of Caspase-1(Cleaved Caspase-1)and the active fragment of gas⁃dermin D N(GSDMD-N)in the PBMCs were detected by Western blotting.The expression of miR-664b-3p was detected by qPCR.The protein expression of serum interleukin-1β(IL-1β)and interleukin-18(IL-18)was detected by ELISA.Target scan 7.2 software was used to predict whether there were complementary binding sites between miR-664b-3p and Caspase-1 mRNA 3′UTR,and the wild-type and mutant Caspase-1 mRNA 3′UTR plasmids containing binding sites were constructed,respectively,and the effect of miR-664b-3p on the luciferase reporter gene activity was detected by double lu⁃ciferase reporter gene system.Results The expression of Cleaved Caspase-1,GSDMD-N,IL-1β,and IL-18 proteins was higher and miR-664b-3p expression was lower in PBMC of the DVT group than in the healthy control group(all P<0.05).Bioinformatics analysis showed that there were complementary binding sites between miR-664b-3p and 3′UTR of Caspase-1 mRNA;transfection of miR-664b-3p mimics could reduce the activity of 3′UTR luciferase reporter gene of wildtype Caspase-1 mRNA(P<0.05),but had no significant effect on the activity of 3′UTR reporter gene of mutant Caspase-1 mRNA(P>0.05).Conclusion The expression of miR-664b-3p in PBMC of DVT patients decreased,and then the inhi⁃bition on the expression of its target gene Caspase-1 was released;elevated Caspase-1 activity significantly increased the expression of GSDMD-N protein,which resulted in pyroptosis and increased expression of IL-1βand IL-18,and ultimately l

关 键 词：深静脉血栓形成 细胞焦亡 微小RNA-664b-3p 半胱氨酸天冬氨酸蛋白水解酶1 焦孔素D 白细胞介素 

分 类 号：R392[医药卫生—免疫学]