miR-142-3p 靶向ATG4c 调控RAW264.7 巨噬细胞自噬

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、50 ng/ ml 雷帕霉素作用2 h、EBSS 饥饿作用12 h、10 nmol/ L 的3-甲基腺嘌呤(3-MA)作用12 h 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LC3域的相对表达。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忆-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LC3域蛋白表达显著下调,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。

miR-142-3p regulates autophagy by targeting ATG4c in RAW264.7 macrophages

Objective:To study the mechanism of miR-142-3p regulated Autophagy Association Gene (ATG4c),to explore autophagy pathway in RAW264.7 macrophages.Methods: To predict the target genes of miR-142-3p by Bioinformatics Software-ATG4c.Then to establish pMIR-Report-ATG4c and pMIR-Report-ATG4c mut recombinant plasmid,in order to confirm the regulatory relation between miR-142-3p and ATG4c through dual luciferase assay,qRT-PCR,Western blot.Four groups was assigned by different treatment,they were normal cells control group,2 h 50 ng/ ml rapamycin treated group,12 h EBSS hunger treated group and 12 h 10 nmol/ L 3-methyl adenine (3-MA) treated group,to detect miR-142-3p relative expression in RAW264.7 cell line by quantitative Real- Time PCR (qRT-PCR).MiR-142-3p mimics,miR-142-3p inhibitor and miR-142-3p control was transfected in RAW264.7 cells respectively,the relative expression of miR-142-3p and LC3 expression was examined by qRT-PCR and Western blot.Results: It revealed the target gene (ATG4c) of miR-142-3p by Dual luciferase assay,qRT-PCR and Western blot;Compared with the control group,the expression of miR-142-3p was obviously up-reguated from the rapamycin and hunger treated groups in RAW264.7cell line,on the contrary,miR-142-3p was obviously down regulated from the 3-MA treated group in RAW264.7 cell line;meanwhile,compared with the control group,LC3 expression was significantly lower from miR-142-3p mimics group;however,LC3 expression was significantly higher from miR-142-3p inhibitor group.Conclusion: MiR-142-3p can target associated genes ATG4c to regulate autophagy in RAW264.7 cell line.