miR-142-3p靶向ATG4c调控RAW264.7巨噬细胞自噬

发布时间：2018-04-28 09:52

  本文选题：miR--p + 细胞自噬　； 参考：《中国免疫学杂志》2016年11期

【摘要】：目的:研究miR-142-3p对自噬相关基因ATG4c的靶向调控作用,探究miR-142-3p影响RAW264.7细胞自噬途径的作用机制。方法:生物信息学软件分析miR-142-3p的靶基因为ATG4c,构建pMIR-Report-ATG4c和pMIR-Report-ATG4c mut重组质粒,双荧光素酶报告系统、qRT-PCR、Western blot验证miR-142-3p与ATG4c的靶向作用;将做不同处理的RAW264.7细胞分为4组:正常细胞作为对照、50ng/ml雷帕霉素作用2h、EBSS饥饿作用12 h、10 nmol/L的3-甲基腺嘌呤(3-MA)作用12h后,实时荧光定量PCR(qRT-PCR)检测miR-142-3p不同干预组中的相对表达情况;将miR-142-3p mimics、miR-142-3p inhibitor及miR-142-3p control分别转染到RAW264.7细胞中,检测miR-142-3p和LC3Ⅱ的相对表达。结果:双荧光素酶报告系统、qRT-PCR、Western blot验证miR-142-3p通过靶向作用于ATG4c的3'-UTR抑制其表达;与对照组相比,雷帕霉素和饥饿处理的RAW264.7细胞miR-142-3p明显上调,而3-MA处理组miR-142-3p明显下调;与miR-142-3p control组相比,转染miR-142-3p mimics组中LC3Ⅱ蛋白表达显著下调,而miR-142-3p inhibitor组中表达显著上调。结论:miR-142-3p通过靶向调控自噬相关基因ATG4c,参与RAW264.7小鼠巨噬细胞自噬的调控。
[Abstract]:Aim: to investigate the targeted regulation of miR-142-3p on autophagy related gene ATG4c and to explore the mechanism of miR-142-3p affecting autophagy pathway in RAW264.7 cells. Methods: the target base of miR-142-3p was analyzed by bioinformatics software. The recombinant plasmids of pMIR-Report-ATG4c and pMIR-Report-ATG4c mut were constructed. The double luciferase report system was used to detect the targeting effect of miR-142-3p and ATG4c by Western blot. The RAW264.7 cells treated with different treatments were divided into four groups: normal cells were treated with 50ng / ml rapamycin for 2 h and then treated with 3-methyladenine 3-MAfor 12 h or 10 nmol/L. The relative expression of miR-142-3p in different intervention groups was detected by real-time quantitative PCR qRT-PCRR. MiR-142-3p mimicstmiR-142-3p inhibitor and miR-142-3p control were transfected into RAW264.7 cells respectively to detect the relative expression of miR-142-3p and LC3 鈪，

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