miR-148a-3p通过抑制IL-12/IL-12Rβ_(1)/IL-12Rβ_(2)/IFN-γ通路抑制Th1细胞极化  

miR-148a-3p inhibits Th1 cell polarization via down-regulating IL-12/IL-12Rβ_(1)/IL-12Rβ_(2)/IFN-γpathway

作　　者：张晗[1,2] 李雪 刘元林 刘伟江 王洋 白海涛 张金霞 苏菲娅 袁福临 李露 孟广雨 郑荣秀 张毅[2] ZHANG Han;LI Xue;LIU Yuan-lin;LIU Wei-jiang;WANG Yang;BAI Hai-tao;ZHANG Jin-xia;SU Fei-ya;YUAN Fu-lin;LI Lu;MENG Guang-yu;ZHENG Rong-xiu;ZHANG Yi(Department of Pediatrics,General Hospital of Tianjin Medical University,Tianjin 300052,China;Institute of Radiation Medicine,Academy of Military Medical Sciences,Beijing 100850,China)

机构地区：[1]天津医科大学总医院儿科,天津300052 [2]军事科学院军事医学研究院辐射医学研究所,北京100850

出　　处：《中国药理学与毒理学杂志》2021年第11期816-823,共8页Chinese Journal of Pharmacology and Toxicology

基　　金：国家重点研发计划(2016YFC1000305);天津市卫生行业重点攻关项目(16KG123);天津市自然科学基金重点项目(17JCZDJ36400);天津市科技局科学技术普及项目(18KPHDSF00140)。

摘　　要：目的探讨miR-148a-3p调控1型辅助性T(Th1)细胞极化调控的分子机制。方法分离6周龄小鼠的脾单个核细胞,经免疫磁珠阴性分选获得初始CD4+(naïveCD4+)Th细胞,用流式细胞术测定naïve CD4+Th细胞CD62L表达水平。用小鼠白细胞介素12(IL-12)和IL-2及抗小鼠IL-4,CD28和CD3ε单克隆抗体的混合因子体外诱导naïveCD4+Th细胞向Th1细胞极化,同时在诱导体系中加入miR-148a-3p模拟物,诱导72 h。流式细胞术检测干扰素γ(IFN-γ)和CD4双阳性(IFN-γ+CD4+)细胞百分比,实时荧光定量PCR检测Th1细胞极化相关基因IFN-γ、信号转导与转录激活因子1(STAT1)、IL-12受体β_(1)(IL-12Rβ_(1))、IL-12Rβ_(2)、STAT4和T细胞介导的转录调节因子21(Tbx21)mRNA表达水平;Western印迹法检测Th1细胞极化关键转录因子STAT4和磷酸化STAT4(p⁃STAT4)蛋白表达水平。经生物信息学分析预测miR-148a-3p对IL-12/IL-12Rβ_(1)/IL-12Rβ_(2)/IFN-γ通路分子IL-12Rβ_(1),IL-12Rβ_(2),Tbx21和STAT4的调控靶标序列,用双荧光素酶载体psiCHECK2分别构建IL-12Rβ_(1),IL-12Rβ_(2),Tbx21和STAT4的3′UTR序列(psi-wt)及靶标点突变(psi-mutant)序列的克隆载体。将psi-wt和psi-mutant载体分别与miR-148a-3p模拟物共转染至人宫颈癌HeLa细胞,转染36 h后裂解细胞进行双荧光素酶报告基因检测。结果经免疫磁珠分选获得的naïveCD4+Th细胞高表达CD62L,表明naïveCD4+Th细胞分离成功;诱导后Th1细胞极化百分比为(71±5)%,miR-148a-3p模拟物转染后Th1细胞极化百分比下降至(61±3)%(P<0.05)。miR-148a-3p模拟物转染可下调Th1极化关键转录因子STAT1(P<0.01),Tbx21(P<0.01)和STAT4(P<0.01)mRNA表达水平,同时IL-12/IL-12Rβ_(1)/IL-12Rβ_(2)/IFN-γ通路的关键受体IL-12Rβ_(1)(P<0.05)和IL-12Rβ_(2)(P<0.01)及调控Th1极化关键细胞因子IFN-γ(P<0.01)mRNA表达水平亦显著下调。miR-148a-3p模拟物转染可显著下调Th1极化过程所必需的STAT4和p-STAT4蛋白表达水平。生物信息学预测miR-148a-3p靶OBJECTIVE To explore the molecular mechanism of miR-148a-3p in regulating type 1 helper T(Th1)cell polarization.METHODS The untouched naïve CD4+Th cells were isolated from splenic mononuclear cells of 6-week-old mice via negative CD4 immunomagnetic bead isolation kit,and were induced to polarize into Th1 cells by interleukin-12(IL-12),IL-2,anti-IL-4 antibody,anti-CD28 antibody and anti-CD3εantibody stimulating(normal induction).miR-148a-3p mimic was added into the co-culture system and flow cytometry was used to detect the percentage of interferon-γ(IFN-γ)+CD4+Th cells after 72 h induction.The expression levels of Th1 polarization related genes IFN-γ,signal transducer and activator of transcription 1(STAT1),IL-12 receptor subunitβ_(1)(IL-12Rβ_(1)),IL-12Rβ2,STAT4 and T-box transcription factor 21(Tbx21)were measured via quantitative real-time PCR.Western blotting was applied to analyze the protein level of STAT4 and phosphorylated STAT4(p-STAT4),the key transcription factors during Th1 cell polarization.The target sequence of miR-148a-3p regulating the expression of IL-12Rβ_(1),IL-12Rβ2,Tbx21 and STAT4 were identified by bioinformatic analysis.The 3'UTR sequence(psi-wt)and target mutant sequence(psi-mutant)of IL-12Rβ_(1),IL-12Rβ2,Tbx21 and STAT4 were cloned by dual luciferase vector psiCHECK2,respectively.The psi-wt and psi-mutant vector were transfected into HeLa cells with miR-148a-3p mimic.After 36 h transfection,the cells were lysed for double luciferase reporter gene detection.RESULTS The untouched naïve CD4+Th cells isolated from splenic mononuclear cells showed high-level expression of CD62L.Compared with normal induction group,the percentage of Th1 cell polarization in miR-148a-3p mimic treated group was decreased from(71±5)%to(61±3)%(P<0.05).miR-148a-3p down-regulated the expression of IFN-γ(P<0.01),STAT1(P<0.01),Tbx21(P<0.05)and STAT4(P<0.01).The expression levels of IL-12Rβ_(1)(P<0.05)and IL-12Rβ2(P<0.01),receptors of IL-12/IL-12Rβ_(1)/IL-12Rβ2/IFN-γpathway,were also significantly d

关 键 词：miR-148a-3p 1型辅助性T细胞 细胞极化 干扰素Γ 

分 类 号：R991[医药卫生—毒理学]