pSicoR/miR-17-5p靶向调控UNC-51样激酶1(ULK1)及其对细胞自噬的影响

Construction of Recombined p Sico R/miR-17-5p shRNA Lentivirus and Its Effects on ULK1 Expression and autophagy in RAW264.7 Cells

目的为了探讨miR-17-5p对自噬相关基因ULK1的调控作用及其对细胞自噬的影响,为更深入研究miR-17-5p调控细胞自噬机制建立理论依据。方法构建p Sico R-miR-17-5p过表达重组质粒,包装miR-17-5p过表达慢病毒,构建ULK1及其突变体ULK1-mut的pmiR-report载体,通过双荧光素酶报告系统验证miR-17-5p对ULK1的靶向关系,利用Western blot检测ULK1与LC3B蛋白的表达量。结果成功构建p Sico R-miR-17-5p过表达重组质粒并且成功将ULK1及其突变体ULK1-mut的3′-UTR插入到pmiR-report载体中;双荧光素酶报告系统结果表明转染miR-17-5p及ULK1-3′-UTR,miR-17-5p过表达组相比对照组相对荧光强度降低1.9倍(P<0.05),说明miR-17-5p对ULK1的表达有抑制作用,而转染miR-17-5p及mut-ULK1-3′-UTR之后检测显示相对荧光强度并没有降低,说明miR-17-5p是靶向作用于ULK1,并对其进行负调控;Western blot显示miR-17-5p抑制ULK1蛋白的表达,并且抑制LC3II/I蛋白的表达。结论 miR-17-5p直接靶向作用于自噬相关基因ULK1,并且对其负调控,抑制细胞自噬的发生。

Objective To construct the lentiviral vector which can over-expression miR-17-5p and explore its targeted effects on ULK1. For further study the role of miR-17-5p on the mechanism of autophagy. Methods Based on chemical technology to synthetic stem-loop structure RNA of miR-17-5p, and cloned it into the linearized p Sico R plasmid which was digested by restriction enzyme and sequenced, the positive recombinants were transfected into HEK-293 T cells; To validate the targeted regulatory relationship between miR-17-5p and ULK1 3'-UTR through the relative luciferase activity, Western blot and real-time PCR test. Using transmission electron microscope to observe the formation of autophagosome and then to determine the occurrence of autophagy. Results The PCR, restriction enzyme digestion analysis and DNA sequencing demonstrated that the recombinant plasmids of p Sico R/miR-17-5p and Report-ULK1 3'-UTR were constructed successfully, and confirmed that the over-expression of miR-17-5p suppressed the expression level of ULK1 protein significantly(P<0.05); suppression of miR-17-5p expression increased the expression level of ULK1 protein significantly(P<0.05). Further more,over-expression of miR-17-5p suppressed the occurs of cell autophagy. Conclusion The recombinant plasmid of p Sico R/miR-17-5p has been constructed successfully, demonstrating that miR-17-5p can directly target on the gene expression of ULK1. It is negatively regulated at the post-transcriptional level, construct the foundation of miRNA for regulation of the further research on autophagy.